Praneeth Kuninty was a PhD Student in the research group Biomaterials Science and Technology, his supervisors were Professor Gerrit Storm and Dr. Jai Prakash from the Faculty of Science and Technology (TNW)
Pancreatic cancer is the most aggressive tumor type with a 5-year overall survival of only 8% and the median survival time is about 6 months. Chemotherapy is the only option for patients with advanced pancreatic cancer, as only less than 20% tumors are resectable. The progression of pancreatic cancer is mainly supported by abundant fibrosis or desmoplasia or tumor stroma. In tumor stroma, cancer-associated fibroblasts (CAFs) mostly originate from pancreatic stellate cells (PSCs), which are shown to elicit tumor promoting activities by secreting different cytokines and growth factors. PSCs produce the abundant extracellular matrix in the tumor stroma, which acts as a physical barrier for drug delivery to the tumor tissue. PSCs interact closely with tumor cells to create a tumor-promoting microenvironment that facilitates tumor growth, angiogenesis, and metastasis, which indicates a role for PSCs in tumor progression. In this thesis, we, therefore, aim to develop novel therapeutic strategies to inhibit the pro-tumorigenic functions of PSCs.
Chapter 1 is a general introduction of pancreatic tumor stroma to the work presented in this thesis. Given the breadth of pancreatic cancer biology, it is not possible to cover all the topics in this brief introductory chapter. Therefore, in different sections, some of the themes relevant to the studies reported in this are discussed. In the first few sections, epidemiology and treatment options are discussed. In another section, the tumor microenvironment concept and therapeutic strategies to target tumor stroma have been discussed. Moreover, the outline and scope of this thesis are discussed.
In chapter 2, we introduce the general concept of miRNA deregulation in different cell types of tumor microenvironment and their significance on tumor progression and metastasis. Shortly, we discussed the strategies of miRNA delivery to target stromal cells within the tumor microenvironment.
In chapter 3 we focused on the identification of novel miRNA (199a/-214) therapeutic targets in pancreatic CAFs from patients and in human primary PSCs. In this study, we investigated their therapeutic role by studying the impact of inhibition of these miRNAs using anti-miRNAs in PSCs differentiation, growth and migration as well as PSC-induced pro-tumorigenic processes. Overall, this study signifies miRNAs as key therapeutic targets in regulating PSC activation in pancreatic cancer.
Naked miRNAs are unstable, hydrophilic, negatively charged and high molecular weight and these properties prevent them from crossing cell membrane. To overcome these hurdles, in chapter 4 we designed and developed a novel peptide-based nanocomplexes for the delivery of anti-miRNA oligonucleotides into PSCs. Here, we showed that delivery system of miRNAs into PSCs led to the inhibition of differentiation markers and also inhibited the PSC-mediated tumor growth and size of heterospheriods which comprised of PSCs and tumor cells. Altogether, our novel anti-miRNA delivery system has higher uptake in PSCs, which can inhibit PSC-mediated protumorigenic activity.
Next to miRNA, we designed novel strategies to block the identified targets and examined their inhibitory effects in vitro and in vivo. In chapter 5, we identified the new surface target (Integrin α5/ ITGA5), which is highly expressed in pancreatic cancer patients in relation to stroma and found that the overexpression of ITGA5 was linked to significant poor disease-free survival and overall survival. Then, we explored ITGA5 for its role in primary PSCs reprogramming by knocking-down ITGA5 with shRNA in vitro. In vivo, we studied the effect of ITGA5 on tumor growth and tumors derived from co-injection of Panc-1 and ITGA5-knockdown (KD) PSCs had slower tumor growth compared to that of Panc-1 and control shRNA PSCs. These findings suggest that ITGA5 in PSCs plays a crucial role in maintaining their phenotype and inducing their pro-tumorigenic actions.
Having identified ITGA5 as a key target in vivo, we aimed to achieve effective blocking of ITGA5 pharmacologically for future (pre)-clinical applications. In chapter 6, we designed a novel integrin binding peptide (so-called AV3) against the ITGA5 receptor. Then, we investigated the therapeutic effects of AV3 by administering either in co-injection or clinically-relevant PDx tumor models for the treatment of the pancreatic tumor. In PDx study, in combination with AV3 and chemotherapy reduced tumor growth in mice by 80% while chemotherapy showed moderate effects. These data indicate that we have been able to break down the physical barrier of the tumor stroma for the drug penetration.
This thesis focused on PSCs, key cells in a pancreatic tumor to induce tumor growth, and identified novel targets which were miRNA-199a and 214 and ITGA5. These targets were proven to be key therapeutic targets. We showed that inhibition of these targets either by anti-miRNA or a peptide (AV3), respectively, leads to inhibition of PSC reprogramming and thereby their pro-tumorigenic effects in vitro and/or in vivo.