Biology subjects

Marafioti, T.

Publications and source records attributed to Marafioti, T..

9 recordsLinked to original sources

bioRxiv2023

Mitochondrial dsRNA from B-ALL cells stimulates mesenchymal stromal cells to become cancer associated fibroblasts

Cancer associated fibroblasts (CAF) arising from bone marrow-derived mesenchymal stromal cells (MSC) are prominent in B-precursor acute lymphoblastic leukaemia (B-ALL). We have previously shown that CAF formation is triggered by exposure to reactive oxygen species-inducing chemotherapy and that CAF support chemoresistance by donating mitochondria to the cancer cells, through tunnelling nanotubes. In the present study, we show that exposure of MSC to ALL cell lines, PDX and primary cells or their conditioned media can also trigger CAF formation, in an oncogene-dependent manner. Using bulk RNA sequencing in cell lines, we show that the MSC to CAF transition is accompanied by a robust interferon pathway response and we have validated this finding in primary cells. Using confocal microscopy and flow cytometry, we identify the take up of leukaemia cell-derived mitochondrial dsRNA by MSC as a proximate trigger for the MSC to CAF transition. We show that degradation of dsRNA in ALL cell conditioned media by DMSO ablates the ability of the conditioned media to stimulate MSC to CAF transition. Since we find that only specific primary driver genetic subtypes of B-ALL possess the property to directly generate CAFs, we propose this phenomenon as the first mechanistic insight into the strong relationship between acute lymphoblastic leukaemia genetic subtype and survival outcomes. Key pointsX Exposure of MSC to B-precursor ALL cell lines triggers cancer-associated fibroblast formation in an oncogene-dependent manner The proximate trigger for CAF formation is ALL-derived mitochondrial double stranded RNA

cancer biology↗

bioRxiv2023

Phenotyping of lymphoproliferative tumours generated in xenografts of non-small cell lung cancer

Patient-derived xenograft (PDX) models involve the engraftment of tumour tissue in immunocompromised mice and represent an important pre-clinixtcal oncology research. A limitation of non-small cell lung cancer (NSCLC) PDX model derivation in NOD-scid IL2Rgammanull (NSG) mice is that a subset of initial engraftments are of lymphocytic, rather than tumour origin. In the lung TRACERx PDX pipeline, lymphoproliferations occurred in 17.8% of lung adenocarcinoma and 10% of lung squamous cell carcinoma transplantations, despite none of these patients having a prior or subsequent clinical history of lymphoproliferative disease. Lymphoproliferations were predominantly human CD20+ B cells and had the immunophenotype expected for post-transplantation diffuse large B cell lymphoma. All lymphoproliferations expressed Epstein-Barr-encoded RNAs (EBER). Analysis of immunoglobulin light chain gene rearrangements in three tumours where multiple tumour regions had resulted in lymphoproliferations suggested that each had independent clonal origins. Overall, these data suggest the presence of B cell clones with lymphoproliferative potential within primary NSCLC tumours that are under continuous immune surveillance. Since these cells can be expanded following transplantation into NSG mice, our data highlight the value of quality control measures to identify lymphoproliferations within xenograft pipelines and support the incorporation of strategies to minimise lymphoproliferations during the early stages of xenograft establishment pipelines. To present the histology data herein, we developed a Python-based tool for generating patient-level pathology overview figures from whole-slide image files; PATHOverview is available on GitHub (https://github.com/EpiCENTR-Lab/PATHOverview).

cancer biology↗

bioRxiv2023

Genomic evolution of non-small cell lung cancer patient-derived xenograft models

Patient-derived xenograft (PDX) models of cancer, developed through injection of patient tumour cells into immunocompromised mice, have been widely adopted in preclinical studies, as well as in precision oncology approaches. However, the extent to which PDX models represent the underlying genetic diversity of a patients tumour and the extent of on-going genomic evolution in PDX models are incompletely understood, particularly in the context of heterogeneous cancers such as non-small cell lung cancer (NSCLC). To investigate the depiction of intratumour heterogeneity by PDX models, we derived 47 new subcutaneous multi-region PDX models from 22 patients with primary NSCLC enrolled in the clinical longitudinal cohort study TRACERx. By analysing whole exome sequencing data from primary tumours and PDX models, we find that PDX establishment creates a genomic bottleneck, with 76% of PDX models being derived from a single primary tumour subclone. Despite this, multiple primary tumour subclones were capable of PDX establishment in regional PDX models, indicating that PDX libraries derived from multiple tumour regions can capture intratumour heterogeneity. Acquisition of somatic mutations continued during PDX model expansion, and was associated with APOBEC- or mismatch repair deficiency-induced mutational signatures in a subset of models. Overall, while NSCLC PDX models retain truncal genomic alterations, the absence of subclonal heterogeneity representative of the primary tumour is a major limitation. Our results emphasise the importance of characterising and monitoring intratumour heterogeneity in the context of pre-clinical cancer studies.

cancer biology↗

bioRxiv2019

Megakaryocytes, erythropoietic and granulopoietic cells express CAL2 antibody in myeloproliferative neoplasms carrying CALR gene mutations

The discovery of mutated Calreticulin (CALR) in myeloproliferative neoplasms (MPN) has provided proof of clonality, diagnostic importance, and influence on prognosis of this pathology. The identification of this MPN-associated driver mutation -currently based on molecular assays- is represented as a major diagnostic criterion for essential thrombocythaemia (ET), pre-fibrotic myelofibrosis and primary myelofibrosis (PMF) in the updated World Health Organization (WHO) 2008 classification. In the present study, we validated by immunohistochemistry the diagnostic usefulness of the monoclonal CAL2 antibody. Cases of acute myeloid leukaemia (AML) and myelodysplastic/ myeloproliferative neoplasms (MDS/MPN) have been also investigated to assess the specificity of CAL2 antibody. For this purpose, the result of the CAL2 immunostaining was compared with the result of molecular assays. Additionally, we investigated by double staining whether expression of mutated CALR can also be demonstrated on cells of the erythroid and myeloid lineage. We confirmed the usefulness of the CAL2 monoclonal antibody in successfully detecting mutant CALR in bone marrow biopsies. We showed that the immune-reactivity of CAL2 was absolutely restricted to the presence of CALR mutations, which were seen only in ET and MDS/MPN biopsies, but not in AML biopsies (14/14). There was 100% concordance in biopsy specimens with the concomitant molecular results. We applied double staining technique and confirmed that a subpopulation of granulopoietic and erythropoietic cells express mutated CALR as demonstrated with the CAL2 antibody in cases of MPNs. This supports the suggestion that the CALR mutations occur in a multipotent progenitor capable of generating both myeloid and erythroid progeny with preferential expansion of megakaryocytic cell lineage as a result of CALR mutation in an immature hematopoietic stem cell.

pathology↗

bioRxiv2019

Similar distribution of T-cell subsets in Crohn's disease and in diverticulitis provide evidence against a primary causal role for these cells in Crohn's

Background and AimsT lymphocytes are found in abnormally large numbers in the bowel in Crohns disease. This has led to the assumption by some that these cells play a causal role in the pathogenesis of what has been labelled an autoimmune disease. An alternative explanation for their presence is that, as part of the adaptive immune system, the accumulation of these cells is not a primary phenomenon, but is a secondary adaptive immune response to faecal material in the bowel wall. To distinguish between these two processes we compared the T-cell repertoire in the bowel in Crohns with that in diverticulitis, where the primary pathology is mechanical, with a subsequent immune response to the accumulated faecal material. MethodsSix cases of Crohns disease and six patients with diverticulitis were studied. Dewaxed sections of bowel were stained with Anti-CD4, Anti-CD8, Anti-FOXP3 and Anti-CD25 to identify cytotoxic T-cells, NK-Tcells; T-helper and T-reg T-cells. ResultsNo differences were found in the distribution of the different T-cell markers in either the mucosa or in areas of inflammation in the two conditions. ConclusionThe accumulation of T-lymphocytes in the bowel in Crohns disease is likely to be a sign of an adaptive immune response to faecal material within the bowel rather than an indication of a primary causal immune attack on the bowel that produces the disease.

immunology↗

bioRxiv2019

Immune surveillance in clinical regression of pre-invasive squamous cell lung cancer

Before squamous cell lung cancer develops, pre-cancerous lesions can be found in the airways. From longitudinal monitoring, we know that only half of such lesions become cancer, whereas a third spontaneously regress. While recent studies have described the presence of an active immune response in high-grade lesions, the mechanisms underpinning clinical regression of pre-cancerous lesions remain unknown. Here, we show that host immune surveillance is strongly implicated in lesion regression. Using bronchoscopic biopsies from human subjects, we find that regressive carcinoma in-situ lesions harbour more infiltrating immune cells than those that progress to cancer. Moreover, molecular profiling of these lesions identifies potential immune escape mechanisms specifically in those that progress to cancer: antigen presentation is impaired by genomic and epigenetic changes, TGF-beta signalling is overactive, and the immunomodulator TNFSF9 is downregulated. Changes appear intrinsic to the CIS lesions as the adjacent stroma of progressive and regressive lesions are transcriptomically similar. This study identifies mechanisms by which pre-cancerous lesions evade immune detection during the earliest stages of carcinogenesis and forms a basis for new therapeutic strategies that treat or prevent early stage lung cancer.

cancer biology↗

bioRxiv2019

Exaggerated in vivo IL-17 responses discriminate recall responses in active TB

Host immune responses at the site of Mycobacterium tuberculosis (Mtb) infection serve to contain the pathogen, but also mediate the pathogenesis of tuberculosis (TB) and onward transmission of infection. Interferon gamma (IFN{gamma}) responses do not discriminate between protection and pathogenicity, but IL-17A/F responses, known to drive pathology in diverse chronic inflammatory diseases, have also been associated with TB pathogenesis in animal models. At the site of in vivo immune recall responses to Mtb modelled by the tuberculin skin test, we show for the first time that active TB in humans is also associated with exaggerated IL-17A/F expression, accumulation of Th17 cells and IL-17A/F bioactivity, including increased neutrophil recruitment and matrix metalloproteinase-1 expression directly implicated in TB pathogenesis. These features discriminate recall responses in patients with active TB from those with cured or latent infection and are also evident at the site of TB disease. Our data support targeting of this pathway in host-directed therapy for TB.

immunology↗

bioRxiv2018

Genomic and transcriptomic determinants of therapy resistance and immune landscape evolution during anti-EGFR treatment in colorectal cancer

Anti-epidermal growth factor receptor (EGFR) antibodies (anti-EGFR-Ab) are effective in a subgroup of patients with metastatic colorectal cancer (CRC). We applied genomic and transcriptomic analyses to biopsies from 35 RAS wild-type CRCs treated with the anti-EGFR-Ab cetuximab in a prospective trial to interrogate the molecular resistance landscape. This validated transcriptomic CRC-subtypes as predictors of cetuximab benefit; identified novel associations of NF1-inactivation and non-canonical RAS/RAF-aberrations with primary progression; and of FGF10- and non-canonical BRAF-aberrations with AR. No genetic resistance drivers were detected in 64% of AR biopsies. The majority of these had switched from the cetuximab-sensitive CMS2-subtype pretreatment to the fibroblast- and growth factor-rich CMS4-subtype at progression. Fibroblast supernatant conferred cetuximab resistance in vitro, together supporting subtype-switching as a novel mechanism of AR. Cytotoxic immune infiltrates and immune-checkpoint expression increased following cetuximab responses, potentially providing opportunities to treat CRCs with molecularly heterogeneous AR with immunotherapy.

cancer biology↗

bioRxiv2018

Pan-cancer deconvolution of cellular composition identifies molecular correlates of antitumour immunity and checkpoint blockade response

The nature and extent of immune cell infiltration into solid tumours are key determinants of therapeutic response. Here, using a novel DNA methylation-based approach to tumour cell fraction deconvolution, we report the integrated analysis of tumour composition and genomics across a wide spectrum of solid cancers. Initially studying head and neck squamous cell carcinoma, we identify two distinct tumour subgroups: immune hot and immune cold, which display differing prognosis, mutation burden, cytokine signalling, cytolytic activity, and oncogenic driver events. We demonstrate the existence of such tumour subgroups pan-cancer, link clonal-neoantigen burden to hot tumours, and show that transcriptional signatures of hot tumours are selectively engaged in immunotherapy responders. We also find that treatment-naive hot tumours are markedly enriched for known immune-resistance genomic alterations and define a catalogue of novel and known mediators of active antitumour immunity, deriving biomarkers and potential targets for precision immunotherapy.

cancer biology↗