PhD Project: Predictive and Functional Evaluation of Patients Derived Tumor Spheroids and Organoids-Investigating the Relationship between Size and Testing Performance

Abstract

Eslam Saleh Sharkawy

Pancreatic Cancer has proven to be one of the most severe forms of cancer with the worst anticipated prognosis. Mortal- ity due to pancreatic cancer is expected to surpass that of breast and colorectal cancer combined by 2030 due to several reasons including aging, advanced stage when pancreatic cancer is detected in patients, lack of effective treatment strat- egy particularly in Pancreatic Ductal Adeno Carcinoma PDAC cases [01]. PDAC contributes to more than 7 % of cancer associated deaths world-wide with an overall survival rate 8.5% for 6-11 months only in patients with advanced stages of the Disease [02]. Pathological, genomic and clinical studies on PDAC patients have shown three different progressive forms of the disease Pancreatic Intraepithelial Neoplasia PanINs, Intraductal Mucinous Papillary Neoplas IPMN and Pancreatic Mucinous Cystic Neoplasm MCN which all express different stages of epithelial dysplasia, lobulocentric atrophy and ductal metaplasia [03].

Treatment options for PDAC sounds to be very limited and mostly include combined chemotherapy with gemcitabine/ nab paclitaxel together with surgical interference. Activation of KRAS oncogene via single point mutation in codon 12, inactivation of tumor suppressor genes CDKN2A, TP53, SMAD4 and R1 and RII receptors of TGF-B and activation of certain signaling pathways that include NFKB, STAT3 and SRC were found to be characteristic for PanINs and its progression to PDAC [04, 05]. Patient-derived xenografts PDXs have been validated as an effective tool to study PDAC but it required large amount of tissues and longer time to generate [06, 07]. However, Genetically Engineered Mouse Models GEMMs were found to be more precisely recapitulating the patho-physiological features of human PDA across different stages of the disease progression [08]. Previous studies have shown that both human PDA and GEMMs share common histological features including extensive stromal structure, diminished vasculature infiltration and less neo- plastic cellularity content. This makes PDA patients-derived organoids and spheroids more difficult to culture as limited amount of epithelium-derived cancer cells are available to isolate. While 2D culture conditions couldn't support the growth of normal or untransformed non-neoplastic pancreatic cells, 3D culture was found to support both normal and transformed cells but otherwise allowed minimal propagation [09]. In this research, we isolate and culture 3D organoids from normal and malignant murine pancreatic tissues from wild type mice(n=10) and PDA GEMMS (n=20) respectively in order to study and model PDA Pathogenesis in Murine Model. Previous studies have shown success rate of around 70-73%. At the same time, Organoids are also planned to be established from non-PDAC tumors e.g. Pancreatic Acinar Cell Carcinoma and Distal Cholangiocarcinoma as the organoleptic and histopathological features of the lesions may indicate. In addition, some tissue samples are taken from the adjacent normal epithelial layers to the site of tumor in order to allow comparison between normal and cancer tissue cells from the same mouse.

Likewise, 3D organoids are planned to be developed from normal (n=10) and malignant human pancreatic tissues (n=40) either sampled during surgical resection or from endoscopic biopsy samples. Then, orthotopic transplantation of the well-established murine and human PDA organoids will be made in wild mice C57BL/6J in order to assess how pathological lesions reminiscent of Pancreatic Intra-epithelial Neoplasia PanINs can be progressed into varying degrees of invasive PDA and may further develop into metastatic carcinomas in other organs e.g. liver, diaphragm, kidney and peritoneum.

After modeling PDA pathogenesis using patient-derived organoid models in both murine and human, characteristic molecular genes signature in each isolated and in-vitro cultured organoid derived from both models will be further char-acterized using Whole Genome Sequencing of over 2000 PDA associated genes [03]. This will be further correlating to the whole transcriptomic analysis and comparative genes expression profiling using bulk RNA seq and q-RT PCR in addition to high-throughput proteomics using Immune-blotting and Immune- histochemistry IHC approaches. This can help investigate whether patients-derived organoids may function as an optimal model that recapitulates the char- acteristics of pancreatic malignancy when comparing histopathological lesions to the corresponding multi-omics data sets of PDAs in human and murine models.

Personalized cancer treatment has proven to be the most effective method as judged by clinicians as some targeted therapies were found to be suitable for only subsets of patients than others who may develop less sensitivity to higher degrees of resistance to the same therapy [03]. In order to investigate how pancreatic organoids may assist clinicians finding the most appropriate target therapy, the isolated 50 PDO organoids including at least 10 organoids derived from normal issues and 40 organoids from tumor tissues will be exposed to a number of candidate anti-cancer com- pounds belonging to 10 categories of chemotherapy which vary according to their mechanism of action. In order to assess the response of PDA organoids to these drugs, two methods including The Cell Viability Assay developed by Promega Bio Sciences and The Electric Impedance Measurement developed by the Heinz-Nixdorf Lehrustul for Bio- medical Electronics at The Technical University of Munich will be comparatively assessed [10-12]. The result will be finally compared to the corresponding patients' clinical data and actual patients' response to medical treatment as suggested by the physician.

Resistance to the most commercially available anti-cancer drugs has been frequently noted by clinicians and it con- stitutes an actual hurdle towards proceeding with an effective treatment strategy despite being precisely personalized [13]. In order to assess how heterogeneous cells in the patients-derived PDOs may respond differently to anticancer drug compounds, we are going to assess the earliest Drug Tolerant Persisters ( DT) and the Extended Drug Tolerant Persisters (EDT) cells isolated from the Murine and Human PDOs before and after exposure to some selected anti-can- cer therapeutics using Single Cell RNA- seq called Drop-seq and then we are going to analyze the obtained data using a bioinformatics platform called Uniform Manifold Approximation and Projection (UMAP) Dimensionality Reduc- tion for further characterizing and clustering persistent cells against different increasing drug concentrations over time.

Furthermore, previous studies have shown that resistance mechanisms of PDAC to anti- cancer therapeutics could be more likely associated with the role of epigenetic dysregulation of apoptotic pathways being one of the intrinsic tumor factors rather than being associated with the effectivity of therapeutic agents themselves (14-16). In order to investi- gate this further, we are going to analyze DT cells and EDT persister cells from both murine and human PDOs using Chromatin Immune-precipitation Technology Chip-Seq with specific antibodies targeted against the histone proteins H3K4me3 and H3K9me3 which were found to activate downstream signaling pathways responsible for attaining the apoptosis-resistance phenotypes of PDAC after treatment.

PDF