Small intestinal neuroendocrine tumours - Disease models, tumour development, and remedy

Sammanfattning: Small intestinal neuroendocrine tumours (SINETs) are malignant neoplasms which at the time of diagnosis often present with distant metastasis. The field of SINET research faces several challenges. There is a lack of preclinical models for studying SINETs, and it is unclear how well currently available models actually recapitulate the tumour disease. The genetic changes that underlie SINET tumour development are largely unknown and, lastly, curative therapy is rarely achieved. Novel therapies, such as the recently FDA-approved 177Lu-octreotate therapy and up-and-coming immunotherapies need to be further investigated to deliver better response rates for SINET patients. In our first two papers (papers I and II), we sought to evaluate frequently used and readily available gastroenteropancreatic neuroendocrine tumour (GEPNET) cell lines as models of neuroendocrine tumour disease. We investigated the characteristics of these cell lines in terms of their neuroendocrine phenotype, genomic background, and therapeutic sensitivity. While several cell lines exhibited an expected neuroendocrine differentiation and harboured genetic alterations characteristic of the GEPNET disease, three cell lines did not. In fact, it turned out that one of the most frequently used cell lines in the field – KRJ-I, together with the cell lines L-STS and H-STS, were incorrectly identified and instead lymphoblastoid cell lines (EBV-immortalised B-lymphocytes). This might have led to the incorrect use and potentially faulty conclusions in a number of GEPNET studies. Among authentic cell lines, we performed a large-scale inhibitor sensitivity screening and predicted that SINETs would be more sensitive to HDACi compared to pancreatic neuroendocrine tumours (PanNET) and PanNET more sensitive to MEKi compared to SINET. The prediction was supported by subsequent experiments with primary tumour cells. In our third paper (paper III), we evaluated a mechanism by which hemizygous loss of SMAD4 could lead to SINET initiation and/or progression by acting as a haploinsufficient tumour suppressor. We found that loss of SMAD4 was associated with a decrease in corresponding mRNA and protein, and that this correlated to patient survival. We also found that the amount of SMAD4 protein in the primary tumour could predict whether the patient presented with distant metastasis. In our last papers (papers IV and V), we investigated the potential for two novel treatment strategies for SINETs. In paper IV we identified an inhibitor, the heat shock protein 90 inhibitor ganetespib, that could synergistically enhance the 177Lu-octreotate therapy for SINETs. Ganetespib was initially found to sensitise SINETs to radiation in a large-scale inhibitor synergy screening, and its radiosensitising effect for radionuclide treatment of SINETs was validated both in mouse xenografts and in primary patient tumours. Lastly, in paper V we characterised the SINET immune microenvironment. Using immunohistochemistry and flow-cytometry we detailed the immune cell composition of the SINET immune microenvironment and could demonstrate the successful isolation and expansion of tumour-infiltrating lymphocytes. We saw that after infiltrating lymphocytes were expanded they could degranulate when challenged with autologous tumour cells. In conclusion, these studies have provided a thorough characterisation of authentic, and provided important information regarding misidentified, frequently used gastroenteropancreatic cell lines. It has also investigated the role of hemizygous SMAD4 loss in the development of SINETs and demonstrated the potential of two novel therapies for SINETs: 177Lu-octreotate combined with Hsp90i ganetespib and immunotherapy.