Colorectal cancer (CRC) is one of most common cancer diseases worldwide. In Swedenapproximately 5,000 new cases of CRC are generated each year, which makes it the thirdmost common cancer disease among both men and women. The past decades ofimproved treatment strategies have considerably increased the five-year survival for CRCpatients. However more could be achieved in this area, in particular for metastatic CRC,which is the cause of most CRC-related deaths. Therefore it is important to study thebiological response to certain treatments induced in CRC to find valuable predictiveand/or prognostic factors to select patients for better suited treatments. The aim of this thesis was to gain insight into the molecular changes that occurfollowing irradiation or treatment with SN-38 in rectal cancer patients or colon cancercell lines by studying the RNA expression, protein expression, DNA cell cycledistribution and apoptotic response. The expression of phosphatase of regenerating liver(PRL) proteins was investigated in rectal cancers from 125 patients included in arandomized clinical trial of preoperative radiotherapy (RT). Increased expression of PRLswas seen at the invasive margin of primary and metastatic cancers compared with theinner area of the tumors. Moreover, strong PRL staining at the invasive margin correlatedto distant recurrence and worse survival of patients in the RT group but not in non-RTgroup (Paper I). Radiosensitivity was studied by treating KM12C, KM12SM andKM12L4a colon cancer cell lines with radiation. KM12C is of low metastatic naturecompared with the highly metastatic KM12SM and KM12L4a. Upregulation of ΔNp73and PRL-3 might contribute to the radioresistant phenotype in KM12C. In contrast,KM12L4a shows a high frequency of apoptosis and lack of upregulation of ΔNp73, PRL-3 and survivin, which might explain its radiosensitive phenotype (Paper II). KM12C,KM12SM and KM12L4a were treated with SN-38 which inhibits topoisomerase 1 (topo-1). The results show that SN-38 induces G2/S arrest and possess the capacity to triggerapoptosis in the three cell lines (Paper III). To further elucidate SN-38 effect on these celllines, the gene expression profile following SN-38 treatment was studied. Oligonucleotidearrays consisting of ~27,000 spots were hybridized with sample and reference cDNA.Both unsupervised and supervised hierarchical clustering analysis, and functional analysiswere performed. Supervised hierarchical clustering gives a strong signal of 1453discriminated genes, the vast majority being upregulated. Both upregulated anddownregulated genes point toward a favorable impact of SN-38 regarding the apoptoticpathways. For example RhoB and Bax are upregulated together with downregulation ofKras and survivin, which promotes apoptosis (Paper IV). In conclusion, PRLs may be valuable biomarkers for RT resistance, predicting apoor prognosis in rectal cancer patients. Targeting radio-resistance factors, such asΔNp73 and survivin may contribute to an increased sensitivity to RT. SN-38 affects cellproliferation and apoptosis.

In this thesis, I used a two-phase gas-liquid segmented microfluidic platform to synthesize drug-loaded block copolymer nanoparticles. In Chapter 2 and 3, the anti-cancer drug 7-ethyl-10-hydroxycamptothecin (SN-38) was physically encapsulated in poly(6-methyl-caprolactone-co-ε-caprolactone)-block-poly(ethylene oxide) (P(MCL-co-CL)-b-PEO) nanoparticles with various drug-to-polymer loading ratios, under different flow conditions. The effects of chemical and flow conditions on the size, morphology, drug loading efficiency, in vitro release and cytotoxicity of the nanoparticles were determined. For various loading ratios, the intermediate total flow rate (Q = 200 µL/min) produced the smallest nanoparticle sizes and pure spheres. The various nanoparticle preparation conditions showed flow-variable release rates and cytotoxicities against MCF-7 cancer cell line. Specifically, we found that release half times of SN-38 from the nanoparticles were from τ1/2 = 0.8 to 3.3 h as the total flow rate increased from Q = 50 to 200 µL/min. We also found that most conditions of SN-38 formulations generated stronger cytotoxicity than free SN-38. As well, at short and intermediate incubation time (48 and 72 h), the cytotoxic potency of microfluidic nanoparticles prepared at Q = 200 µL/min were slightly higher than nanoparticles prepared using a conventional bulk method, while potencies of microfluidic nanoparticles prepared at higher and lower flow rates were slightly lower than the bulk control. In Chapter 4, in order to pursue even higher shear rate and increased throughput, we switched the microfabrication material to silicon/glass from polydimethylsiloxane (PDMS) used in earlier chapters, maintaining the gas-liquid microfluidic reactor design. A comparison between the two microfluidic reactor materials at constant liquid flow rate showed that channel material affected both flow behaviour and the resulting nanoparticle morphologies. A new, single-phase microfluidic strategy was also proposed in order to generate high shear, in which variable high and low shear would arise from periodic changes in channel dimensions. However, issues regarding clogging of the more narrow microchannels require future work of improvements in either reactor design or the microfabrication process. / Graduate / 2019-01-12

Indiana University-Purdue University Indianapolis (IUPUI) / Colorectal cancer is the third most common type of cancer and the third most common cause of cancer-related mortality. There are three types of treatment available to patients, either individually or in combination. Treatments are radiation, chemotherapy, and surgery. In a Phase II clinical trial at IUSM, a multimodality approach was chosen. The patients with locally advanced rectal cancer received preoperative treatment with capecitabine and irinotecan (CPT-11) combination followed by chemoradiation with capecitabine and finally surgery to improve response and decrease local recurrence. Irinotecan and Capecitabine are both prodrugs activated in vivo to SN-38 and 5-FU, respectively. Identification of the molecular markers for 5-FU and Irinotecan efficacy and toxicity is important for the development of more efficient and less toxic treatment strategies for patients with colorectal cancer. The goal of this study was to determine the expression levels of the genes involved in activation and metabolism of capecitabine and irinotecan in pre and post treatment specimens from these patients. The genes quantitated by real-time PCR were carboxylesterase 1 and 2 (CES1 and CES2), thymidylate synthase (TS), β-glucoronidase (β-GUS), thymidine phosphorylase (TP), dihydropyrimidine dehydrogenase (DPD) and topoisomerase I (Topo I). The UGT1A1*28 polymorphism in UDP glucuronosyltransferase 1 is associated with SN-38 toxicity. Therefore, the UGT1A1*28 polymorphism status in patients was determined by PCR-sequencing. Correlative analysis of gene expression and UGT1A1*28 mutation with clinical outcome in this Phase II study was completed.