The challenge of pancreatic cancer therapy and novel treatment strategy using engineered mesenchymal stem cells.

Cancer Gene Therapy (2014) 21, 12–23 & 2014 Nature America, Inc. All rights reserved 0929-1903/14 www.nature.com/cgt

REVIEW

The challenge of pancreatic cancer therapy and novel treatment strategy using engineered mesenchymal stem cells MR Moniri1, L-J Dai1,2 and GL Warnock1 Mesenchymal stem cells (MSCs) have attracted significant attention in cancer research as a result of their accessibility, tumororiented homing capacity, and the feasibility of auto-transplantation. This review provides a comprehensive overview of current challenges in pancreatic cancer therapy, and we propose a novel strategy for using MSCs as means of delivering anticancer genes to the site of pancreas. We aim to provide a practical platform for the development of MSC-based therapy for pancreatic cancer. Cancer Gene Therapy (2014) 21, 12–23; doi:10.1038/cgt.2013.83; published online 3 January 2014 Keywords: pancreatic cancer; mesenchymal stem cells

PANCREATIC CANCER AND TREATMENT SHORTFALL According to 2012 cancer statistics, in the United States pancreatic cancer is ranked 10th in terms of new cancer cases and 4th in cancer deaths,1 with an overall 5-year survival rate of 4% after diagnosis.2 This poor prognosis can largely be attributed to the tendency of this cancer to rapidly spread to the lymphatic system and distant organs.3 Similarly, Canadian cancer statistics (2012) report pancreatic cancer affecting B4600 Canadians each year and with dismal cure rates at about 2–3%.4 The number of newly diagnosed patients nearly equals that of the deceased; no current treatment, whether surgical, chemotherapy or a combination thereof, results in an effective clinical response in more than 20% of patients and no treatment therapies have been found that extend survival beyond a few months. Clinically, pancreatic cancer presents in up to 50% at a locally advanced stage and, in up to 40% with metastatic disease,5 dramatically reducing the chance of curative resection. Factors such as age, smoking, past medical history, and other conditions such as diabetes and pancreatitis have been associated risk factors of pancreatic cancer.6 With the pancreas being both an exocrine and an endocrine organ, it has two distinct functions in the body and thus has different functioning compartmental units. The exocrine component consists of acinar cells, secretory units, and a network of large ducts that drain secretions into the small intestine.7 The acinar cells form the majority of the exocrine tissue and generate digestive enzymes. Ductal cells secrete bicarbonate and mucous.8 The endocrine pancreas consists of clusters of cells commonly known as islets; they are neighboring to the exocrine pancreas and are categorized by their secretory function: b-cells producing insulin, a-cells producing glucagon, d-cells producing somatostatin, PP cells producing pancreatic polypeptide, and finally ecells producing ghrelin.8,9 Both exocrine and endocrine tumors can develop from different pancreatic cell phenotypes.10 Endocrine tumors originate from islet cells and account for 2–4% of newly recognized neoplasms whereas exocrine tumors originating from the ductal tissue are by far the most common form of malignant pancreatic cancer accounting for 95% of newly identified cases.10 Despite the fact that surgical options for pancreatic cancer

are now associated with acceptable outcomes, they often prove ineffective in controlling the disease with reported recurrence rates approaching almost 80% (both locally and distant) and a 5-year survival rate of only 10–24% for cases involving total resection.11–13 Lack of distant metastasis and local vascular invasion are the two criteria that must be present to qualify for a curative resection.3 The most common type of a such resection is known as the Whipple procedure in which the tumor-bearing region of the pancreas along with a portion of stomach, duodenum, gallbladder, and part of bile duct are removed and the remaining regions are reattached to support digestive capabilities of the patient.14 In the case of advanced disease, palliative surgery can be performed to lessen symptoms and pain. Other management options range from systemic chemotherapy alone to combined forms of treatment with chemoradiation and chemotherapy. Chemotherapy treatments can be categorized as adjuvant (treatment after surgery), neo-adjuvant (treatment prior to surgery), and palliative. The most common chemotherapy drugs used to treat pancreatic cancer are the following: gemcitabine (Gemzar), 5-fluouracil (5FU), capecitabine (Xeloda), cisplatin, and oxaliplatin (Eloxatin).15,16 These drugs function on the basis of cross-linking mechanisms in which their reactive region interacts with the cell’s DNA or RNA nucleotides, thus disrupting the cell cycle progression leading to cancer cell apoptosis.17–19 In addition, due to the inability of chemotherapy drugs in distinguishing between normal and cancerous cells, the side effects of these drugs are also detrimental to the normal rapidly dividing cells of the body such as: the cells of the hair follicle and the lining of gut. These drugs are usually administrated individually but can also be used in combination depending on the type and extent of the cancer. However, the survival benefit is limited and many patients with advanced disease are deemed for palliative care. Moreover, these drugs have adverse side effects that cause the patients considerable discomfort while reducing the quality of life. Chemoradiotherapy is an efficient therapeutic approach for pancreatic cancer patients; however, there are no data to support a radiotherapy alone treatment option.3 Finally, given the lack of a unifying chemotherapeutic approach and the numerous

1 Department of Surgery, University of British Columbia, Vancouver BC, Canada and 2Hubei Key Laboratory of Stem Cell Research, Taihe Hospital, Hubei University of Medicine, Shiyan, China. Correspondence: MR Moniri or L-J Dai, Department of Surgery, University of British Columbia, 400-828 West 10th Avenue, Vancouver, BC, Canada V5Z 1L8. E-mail: [email protected] or [email protected] Received 12 November 2013; accepted 9 December 2013; published online 3 January 2014

Pancreatic cancer therapy with mesenchymal stem cells MR Moniri et al

13 debilitating side effects of these drugs/treatments, significant attention has been directed to the tumor microenvironment to identify potential yielding therapeutic targets. Due to these poor treatment options for pancreatic cancer patients, new therapeutic strategies are being developed using anticancer gene therapy agents and treatments as an alternative solution or in combination to the existing treatments. These strategies can be categorized as either using RNA interference or antisense oligonucleotides that inhibits the activated oncogenes (Kirsten rat sarcoma (KRAS), LSM1, Akt, Wnt, etc.) or approaches to restore function of the tumor suppressor genes (p53, p16/ CDKN2A, DPC4/SMAD4, etc.).3,19 Table 1 summarizes the frequency of abnormalities of most common oncogenes and tumor suppressor genes in pancreatic cancer. Two recent very encouraging reports have demonstrated the use of oncolytic poxvirus and lentiviral vectors to successfully treat cancer patients.20,21 Monahan et al.22 describes some of the limitations of using a virus as a vector in a clinical setting. These limitations are summarized as follows: (1) packaging space limitations; (2) increased risk of insertional mutagenesis; (3) the body’s immune system rejection, and (4) limitations of producing high titer virus for clinical use.22 Below, we will briefly discuss some molecular mechanisms involved in pancreatic cancer. Our proposed strategy uses mesenchymal stem cells (MSCs) as vehicles, and regular plasmids as vectors, thereby avoiding virusrelated concerns. In the current study, we focus on combining gene therapy to engineer MSCs with anticancer genes in order to target pancreatic cancer cell lines. Engineered MSCs could serve as an ideal delivery vehicle for anticancer compounds, by homing to cancer cells on the microscopic level. MOLECULAR PATHWAYS AND PANCREATIC CANCER Understanding the molecular mechanisms involved in pancreatic cancer will enable researchers to integrate new solutions for the treatment of this devastating disease. Hanahan et al.23,24 describes the progression of cancer from neoplasia to metastasis in six major steps.23,24 These steps include the following: autonomous production of growth factors, lack of response to antigrowth signals, escaping apoptosis, boundless replicative capability, continuous angiogenesis, and finally tissue invasion/metastasis. There is no particular order for such events to occur and depending on the type of cancer, these events could happen individually, or in combination. It is now widely accepted that among the causes for the progression of this disease, the activation of oncogenes and the inhibition of tumor suppressor genes are the key pivotal elements. Thus, much effort has been dedicated to providing greater understanding about these molecular mechanisms and their role in the progression of the disease. Implications of some of the molecular pathways and anticancer genes that are involved in

Table 1. Genetic profiling of pancreatic carcinoma. Modified from Bhattacharyya19 Common genetic profiling of pancreatic carcinoma Gene Oncogenes KRAS LSM1 AKT Tumor suppressor genes P16/CDKNA2 P53 DPC4/SMAD4 Abbreviation: KRAS, Kirsten rat sarcoma.

& 2014 Nature America, Inc.

Frequency of abnormality (%) 95 87 10–20 490 50–75 55

pancreatic cancer for the current study are discussed in the sections that follow. These pathways were chosen in accordance with the current knowledge on MSC’s antitumorigenic properties and the anticancer genes used in the current study. Pathways of interest for this study are depicted in Figure 1 and explained in depth in the sections that follow. KRAS oncogene and pancreatic cancer Point mutation and gene amplification are two mechanisms by which oncogenes can become activated.3 Almoguera et al.25 reported that more than 90% of human pancreatic adenocarcinomas possess an activation of KRAS viral oncogene homolog.25–27 This is the highest rate of KRAS point mutation observed in any known cancer; however, KRAS mutations are also seen across other models of thyroid (55%), colorectal (35%), and lung (35%) carcinomas.28 KRAS, a 21 kDa protein of Ras family, is known to be associated with cell proliferation and growth.3,19 This guanine nucleotide-binding protein has the ability to automatically inactivate signal transducers. The wild type form of this protein has the ability to switch between the inactive (GDP-bound) and the active (GTP-bound) form of this protein via cell surface receptor initiations.29 However, in the mutated form of KRAS, the oncogenes contain a point mutation substitution of codon 12, 13, or 61.3,28 This mutation leads to the inability of the protein to use its intrinsic downregulation capacity and thus it remains in the active form continuously leading to uncontrolled cell growth. GTP-bound KRAS activates mitogen-activated protein kinase pathway and stimulating gene regulation and cell proliferation. A simplified depiction of this pathway can be observed in Figure 2. The wide scale expression of the KRAS gene in pancreatic carcinoma samples and its role in the cell survival/proliferation mechanisms make it a very significant target for the treatment of pancreatic cancer. PI3K/AKT/mTOR pathway and pancreatic cancer Activation of the phosphatidylinositol 30 -kinase (PI3K-Akt) pathway has been observed in many malignancies. Furthermore, the Akt2 gene has been observed to be amplified in a number of studies including different pancreatic cancer specimens and cell lines.30,31 Akt regulates apoptosis and cell proliferation through multiple mechanisms.32–34 One of the key factors in Ras protein cascade is class 1 PI3K.35 PI3K can phosphorylate negatively charged phospholipids on cell membranes (phosphatidylinositol) that signal the recruitment of pleckstrin homology domain-containing proteins to the cell membrane.32 This recruitment signals the attachment of phosphoinositide-dependent kinase-1 and its target serine/ threonine-specific protein kinase (Akt) to the pleckstrin homology domains, which results in the activation of Akt that is known to be associated with cellular survival pathway.32 On the other hand, the Akt pathway is downregulated by MMAC/PTEN (mutated in multiple advanced cancers/phosphatase on chromosome ten phosphatase) that counteracts to PI3K mechanism by selectively dephosphorylating the negatively charged phospholipid on the cell membrane.36 Inactivation or deletion of the MMAC/PTEN gene has been observed in many tumor models including pancreatic carcinomas.37 It is important to note that many Ras and Akt activators are receptor tyrosine kinases that are also overexpressed in human pancreatic cancer models. These include (but are not limited to) epidermal growth factor receptors, insulinlike growth factor 1 receptors, platelet-derived growth factor receptors, and fibroblast growth factor receptors.36,38–42 Additionally, one of Akt’s main targets is the mammalian target of rapamycin (mTOR), which regulates G1 to S phase in cell cycle progression upon the availability of nutrients.43,44 Phosphorylation of the tuberous sclerosis complex via Akt, activates mTOR.45 Therefore, by activation of PI3K/Akt pathway and availability of nutrients, mTOR becomes activated and could stimulate the Cancer Gene Therapy (2014), 12 – 23


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Figure 1. Molecular pathways involved in pathogenesis and progression of pancreatic cancer. From left to right: with the binding of Wnt to Frizzled receptors, disheveled cascade is activated that in turn activates gene expression that leads to cell proliferation. With the binding of growth factors to the receptor tyrosine kinase, RAS pathway is activated that leads to a series of other molecules activation that can regulate gene expression in the nucleus and further lead to cell proliferation and growth. Similarly, growth factors and survival factors binding to the corresponding receptor can lead to modification of PIP2 and activation of PIP3. This pathway leads to cell proliferation via negative feedback to the mitochondria and activation of gene regulation in nucleus. PTEN can actively inhibit this pathway. Finally, with the binding of TRAIL to its death or decoy receptors cells can regulate apoptosis via the activation of BAX and BAD mitochondria proteins. APAF1, apoptotic proteaseactivating factor 1; BAX, Bcl-2-associated X protein; BCL-2, B-cell lymphoma 2; BID, BH3 interacting-domain death agonist; Caspase, cysteinedependent aspartate-directed proteases; DCR (decoy receptor); DIABLO, direct IAP-binding protein with low pI; DR, death receptor; FADD, Fasassociated protein with death domain; ILK, integrin-linked kinase; MAPK, Mitogen-activated protein kinase; MEKK, MAPK/Erk kinase; MEK, MAPK/Erk kinase; MKK, MAP kinase; mTOR, mammalian target of rapamycin; PIP2, phosphatidylinositol 4,5-bisphosphate, PI3K phosphatidylinositol 3-kinases; PIP3 (phosphatidylinositol (3,4,5)-triphosphate), PTEN (Phosphatase and tensin homolog), PDK1 phosphoinositide-dependent kinase-1, TRAIL, TNF-related apoptosis-inducing ligand); Wnt (wingless type), and TCF (T-cell factors).

translation of proteins needed in the cell cycle progression. Finally, these observations support the proposed importance of the PI3k/ AkT/mTOR pathway as a junction point in survival/death cascades of pancreatic cancer model and their interactions with MSCs (which are known to downregulate the Akt pathway, discussed in section 1.3) which makes understanding the mechanisms involved in this pathway a critical one. Wnt/b-catenin pathway and pancreatic cancer Wnt (Wingless and INT-1 (integration 1)) pathway is known to be associated with the proliferation, morphogenesis, and Cancer Gene Therapy (2014), 12 – 23

differentiation of several organs.46 Furthermore, Wnt is known to be associated with pancreas development.47,48 The role of bcatenin has been reviewed by MacDonald et al.49 There are nineteen different Wnt ligands identified in mammals that are associated with the activation of canonical (b-catenin dependent) and non-canonical (b-catenin independent) pathways.50 Normally b-catenin will become phosphorylated that targets it for degradation. With the binding of Wnt ligand to its receptors of the frizzled51 family of proteins, it enables the activation of an intracellular signaling pathway that transports the b-catenin to the nucleus.50 In the nucleus, b-catenin interacts with transcriptional factors such as T-cell factors and lymphoid enhancer factor and by & 2014 Nature America, Inc.


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Figure 2. Summarizes pros and cons of MSCs within tumor environment. The related mediators are indicated in the corresponded processes. On the left hand side the protumorigenic effects of MSCs are depicted and they can be summarized as: potential malegnancy, immunosuppression, induction of angiogenesis, and EMT. On the right hand, the antitumorigenic effects of MSCs are displayed; potential intrinsic antitumor effects, downregulation of Wnt pathway, and the downregulation of Akt pathway.

this interaction, it can control specific gene expression within the cell (as shown in Figure 1).3 Any mutation leading to the upregulation of Wnt or downregulation of its inhibitors will enhance the function of b-catenin, thus leading to the cells’ proliferation, carcinogenesis, and tumor growth.3 Zeng et al.52 demonstrated that such activation has been observed in over 65% of pancreatic cancers.52 Moreover, the non-canonical Wnts (Wnt signaling that is independent of b-catenin function) have been shown to be deregulated in pancreatic adenocarcinoma samples and could have a significant role in the progression pancreatic cancer.53,54 Finally, in a pancreatic cancer model, the importance of this pathway combined with its interactions with MSCs (discussed in section 1.3) demonstrates that the Wnt pathway is a critical point of study for MSC-related pancreatic cancer therapy. TRAIL and PTEN pathways and pancreatic cancer Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL, also known as Apo2L) is a member of the TNF super family. TRAIL signaling induces a signal that works through the extrinsic apoptotic pathways in the cell. TRAIL was initially identified and cloned55–57 based on its sequence of homology to the extracellular domain of CD95 ligand and TNF. TRAIL is an anticancer protein that selectively causes apoptosis of transformed or tumor cells through the activation of death receptors, with no effects on healthy cells.58 However, gain of TRAIL resistance have been observed in different cancer cells over the years.59 TRAIL messenger RNA is also widely expressed in many tissues including peripheral blood lymphocytes, spleen, thymus, prostate, ovary, small intestine, colon, and placenta that is in contrary to other members of TNF super family of proteins.60 Under normal conditions, TRAIL can contribute to the activation-induced cell death of T cells;61 and the expression of membrane-bound TRAIL in different cells of immune system such as natural killer cells, B cells, monocytes, and dendritic cells post cytokine stimulation can further strengthen their response to tumor tissue.62–66 In humans, TRAIL ligand can bind to five different receptors: TRAIL receptor 1 (DR4), 2 (DR5), 3 (decoy receptor 1, DcR1), 4 (DcR2) and a soluble receptor, osteoprotegerin (OPG).67 The death receptors (leading to cell apoptosis) and the decoy receptors that contain a nonfunctioning truncated death domain have a critical role in cell death homeostasis. In addition, the ligand can bind the OPG receptors but in much lower affinity.68 Mechanisms of TRAIL function are reviewed in depth by Johnstone et al.57 Briefly, following the binding of the ligand to the death receptors, & 2014 Nature America, Inc.

oligomerization of the receptors occur, which in turn recruits FADD (Fas-associated death domains). Fas-associated death domains and caspase 8 combine to produce a functional complex known as death-inducing signaling complex. Next, a cleavage of caspase 8 results in its activation that in turn activates BH3-only protein (BID) and caspase 3 downstream.69,70 BID’s activation leads to a leaky mitochondrial membrane through BAX and BAD (proapoptotic molecules). An apoptosome is formed via an active combination (ATP dependent) of caspase 9, apoptotic protease-activating factor 1 and cytochrome c complex.57 The full mechanism of TRAIL apoptosis pathway is also depicted in Figure 1. Through multiple positive and negative loops, cells would undergo apoptosis and display the morphological features associated with it. The intrinsic apoptotic pathway has a critical role in controlling apoptosis in cancer cells. PTEN (phosphatase and tensin homolog) serves as the main negative regulator of PI3K-AKT-mTOR. Being one of the most commonly activated pathways found in human cancers, PI3K-AKT-mTOR signaling pathway leads to cell growth, survival, and proliferation of cancerous tissue. PTEN dephosphorylates PIP3 to PIP2 thereby directly opposing the activity of PI3K. The mechanisms by which PTEN contributes to apoptosis, loss of cell cycle controls, and genomic mutations that occurs in tumorigenesis are summarized in a review by Liu et al.71 and depicted in Figure 1.71 In addition, PTEN has a vital role in regulating apoptotic stimuli and the response to the chemotherapeutic agents.72 PTEN is one of the tumor suppressor genes most frequently inactivated in human cancers.73 This loss in function is contributed mainly to mutations, deletions, or methylations that occur in the genome of metastatic and de novo human cancers.74 Somatic mutations occur in a large percentage of human cancers, with the highest numbers found in cancerous endometrium (38%), central nervous system (20%), skin (17%), and prostate (14%).75 Genetic mutations in PTEN occur at much lower rates in human pancreatic cancer (B1%);37,75,76 but evidence suggests that mechanisms such as promoter methylation,77 amplified expression of AKT2 genes,78 or lower messenger RNA levels79 could lead to the loss of PTEN function or its corresponding signaling pathways. Different PTEN expressions have been observed in our preliminary analysis of pancreatic cancer cells and thus overexpression of this protein could result in pancreatic cancer cell death via the PTEN/Akt pathway. Moreover, other signaling pathways that are linked to pancreatic cancer such as Ras and growth factors TGF-b and insulin-like growth factor 1, are known to downregulate the expression of PTEN and thus lead to cell proliferation and reduced apoptosis.80–86 Compared with other classical tumor suppressor genes, PTEN is haploinsufficient, thus a single copy is unable to prevent cancer. Improving PTEN function in cancer cells would bring a halt to PTEN mutationdependent cancer cell growth and provide great promise for a cancer therapy option. Pappas et al.87 and Jin et al.88 demonstrated that wild-type PTEN can be introduced into cells by using either viral or non-viral vectors.87,88 Another direct strategy is to deliver cell permeable recombinant wild-type PTEN into cells via fusion PTEN with a cell permeable protein transduction domain such as TAT (trans-activator of transcription).89 In the present work, PTENengineered MSCs will be examined together with TRAIL to investigate a benefit of a dual-targeted therapeutic strategy for treating pancreatic cancer. In summary, there are numerous genes associated with the pathogenesis of pancreatic cancer. Some of those reviewed here were chosen as anticancer genes in combination with MSCs as a means to develop a novel gene therapy strategy for pancreatic cancer. The aim is to induce pancreatic cancer cell apoptosis via targeting both the extrinsic (by activation of proapoptotic receptors or ligands associated with them) and the intrinsic (initiated via mitochondrial pathway as illustrated in Figure 1) apoptotic pathways. Cancer Gene Therapy (2014), 12 – 23


16 MSCS Friedenstein et al.90,91 first identified a group of adult stem cells sourced from human bone marrow, now commonly known as MSCs. MSCs originate from the mesodermal germ layer and are capable of differentiation to tissue of muscle, vascular system, and connective tissue. Bone marrow-derived MSCs’ self-renewal and differentiation capacity allow them to be categorized as stem cells, and their multi-lineage potential is of immense biomedical interest.92,93 Due to their easy extraction from tissue, fast turnover ex vivo, and viability after auto- and allotransplantation, MSCs became the first type of stem cells developed for tissue engineering and regenerative medicine. Aside from their differentiation capacity, these cells could secrete a number of growth factors and cytokines, thus modifying the response of immune system.94,95 Le Blanc et al.96 summarized that MSCs have an intrisnsic capacity to provide important cellular cues in survival of damaged tissues with or without direct contribution in long-term tissue repair.96 Furthermore, Loebinger and Janes97 described the use of stem cells as an ideal anticancer gene delivary vehicle in various preclinical models due to their tumor migration and incorportation capacity.97 In the current study, we aim to use engineered MSCs together with anticancer genes as a delivary vector to treat pancreatic cancer cells. The distribution of MSCs Due to inadequate current understanding of the exact nature and localization of MSCs in vivo, much attention has been devoted to defining the exact nature of their distribution. Aside from bone marrow, MSCs and MSC-like cells are shown to be present in adult and fetal tissues such as pancreas,98–100 circulating blood,101,102 heart,103 adipose tissue,104 skeletal muscle tissue,105 placenta106 and amniotic fluid.107 Vaananen108 suggested that almost all organs that contain connective tissue also contain MSCs.108 The existence of MSCs can be verified using methods such as the use of protein surface biomarkers in vitro109 and post-infusion distribution analysis of MSCs in vivo.110 These methods, although considered to be sensitive, have disadvantages. Using markers could be difficult as they could face the nonspecific engraftment of MSCs in different locations. As an alternative approach, a comparison between characteristics of bone marrow-derived MSCs and MSCs that are isolated from different tissues could increase our understanding in this area. Da Silva Meirelles et al.111 analyzed and characterized MSCs from different tissues and organs of adult mice from the C57Bl/6 and BALB/c mice strains and MSCs were isolated from either perfused or non-perfused animal tissues. They concluded that MSCs can be found throughout all organs and tissues.111 Although MSCs have ubiquitous distribution, bone marrow-derived MSCs are most frequently used in the therapeutic research. Bone marrow-derived MSCs appear as 0.1–5 105 cells (rodents) and 1–20 105 cells (humans) in bone marrow suspensions and are considered a rare population of cells within the bone marrow compartment.112,113 Additionally, Caplan92 describes that the number of MSCs in bone marrow decreases by 20-fold from infancy to the adulthood.114 The classification of MSCs MSCs were first characterized by their clonogenic potential determined by the capacity to configure themselves into colonyforming unit-fibroblast. Different tissue-derived MSCs may have differences in their differentiation capacity even if cultured in identical microenvironments. In a comparative study conducted by Sakaguchi et al,115 isolated cells from bone marrow, synovium, periosteum, skeletal muscle, and adipose tissue were studied for their colony-forming capacity and differentiation into osteogenic and adipogenic cell phenotypes under defined conditions. It was reported that highest rates of osteogenesis were observed in bone marrow-, synovium-, and periosteum- derived MSCs, accordingly. Cancer Gene Therapy (2014), 12 – 23

On the other hand, adipogenesis-positive cells were observed at the highest rate in the synovium- and adipose- derived MSCs. The characteristics of MSCs from the same origin may also vary with the methods of isolation and expansion in different laboratories under different conditions. Much effort has been focused on the direct identification of MSCs from bone marrow and other tissue sources.116–118 Ucelli et al.119 reported on variation of specific markers seen on MSCs in vitro. Negative expression for hematopoietic markers such as CD14, CD34, and CD45 or co-stimulatory molecules of CD80, CD86, and CD40 were observed.119 On the other hand, variable expression levels of CD44, CD71 (transferrin receptor), CD73 (ecto-50 -nucleotidase), CD90 (THY1), CD105 (endoglin), and the ganglioside GD2 and CD271 (low-affinity nerve growth factor receptor) were reported.119 This variability in expression levels may result from species differences, tissue sources, and culture conditions.119 Current acceptable methods for identifying MSCs include a thorough study on the combination of their physical, phenotypic and functional properties.121 The basic criteria that are now widely accepted among scientists for identification of MSCs are summarized by Dominici et al.122 and depicted in Table 2. MSCs in therapeutics MSCs have strong capacity to be differentiated into different tissue types.93,122 Properties of MSCs that have made them attractive cell therapeutic agents include the following: easy and fast acquisition ex vivo; ease of transfection; and their immuneprivileged properties arising from low major histocompatibility complex I and no MHC II expression. However, it is worth mentioning that the expression of MHC I and MHC II in undifferentiated MSCs could be proportionally increased with the differentiation extent of MSCs.123 The therapeutic benefit of MSC transplantation in various disorders characterized by cell injury or cell loss are shown in some recent studies such as myocardial infarction,124,125 traumatic brain injury,126–128 Parkinson’s disease,129 type 1 diabetes,94,130,131 and liver disease.132 Lately, with the discovery of MSCs’ tumor tropism, much interest is dedicated to determining the role MSCs could have in cancer therapy. MSC and cancer Malignant tumor cells live within a complex microenvironment better known as tumor ‘stroma’.133 Some of the complex building blocks found in solid tumors are the supporting cells that include the following: fibroblasts, endothelium, pericytes, lymphatics, and a mononuclear infiltrate.134 These stromal elements have a critical role in tumor survival, structural support, and vascularization. Thus, any therapy targeting both the stromal components and the

Table 2.

Basic criteria for defining human MSCs

Cultivation Phenotype

In vitro differentiation:

Adherence to plastic in standard culture conditions Positive expression (X95%) CD 73 CD 90 CD 105

Negative expression (p2%) CD 14 or CD 11b CD 19 or CD 79a CD 34 CD 45 HLA-DR Under specific stimulus, cells will differentiate into osteoblasts, adipocytes, and chondroblasts

Abbreviation: MSC, mesenchymal stem cell.



17 malignant cells could lead to an efficient anticancer therapeutic approach. Next, we will take a closer look at the properties that MSCs possess that could make them an ideal anticancer agent. Tumor-directed migration and incorporation of MSCs Advancing tumors continuously produce cytokines, chemokines, and other inflammatory mediators.135 Such signals are capable of attracting respondent cell types such as MSCs. Transwell culture studies and animal models in vitro and in vivo were used respectively to determine the tumor tropism capabilities of MSCs. Various studies confirm the capacity of MSCs for tumor tropism and incorporation in almost all human cancer cell lines, such as malignant glioma,136–138 breast cancer,139,140 Kaposi’s sarcomas,141 lung cancer,142 colon carcinoma,140 pancreatic cancer,98,134,143 melanoma144 and ovarian cancer.140 Furthermore, higher frequency of migration and incorporation was seen in in vitro co-cultures and in vivo xenograft tumors, respectively. The mechanism underlying MSC’s tumor tropism is still not fully understood. Production of chemo-attractant molecules from tumors and the expression of corresponding receptors on the MSCs are the required criteria for the migration and tumor tropism of MSCs. Dwyer et al.145 showed a collection of chemokines and cytokines that could interact with MSCs receptors.145 As reviewed by Sasportas et al,136 a number of cytokine-receptor pairs have been found to be associated with the capacity of MSCs to migrate toward tumor tissue.136 These include but are not limited to the following: SDF-1/CXCR4, SCF/c-Kit, HGF/c-Met, vascular endothelial growth factor/vascular endothelial growth factor receptor, MCP/CCR2, and HMGB1/RAGE146–148 and adhesion molecules, 1-and 2-integrins, and L-selectin.146–148 Furthermore, factors such as IL-8, neurotrophin-3, TGF-, IL-1, TNF-a, platelet-derived growth factor, and EGF have also been shown to enhance MSCs tumor tropism capabilities.149,150 In addition, the presence of chemokine receptors such as CCR(1,4,7,9,10), CXCR(4,5,6), CX3CR1, and c-Met on MSCs might be contributing factors to the tumor tropism properties of MSCs.146,151 MSCs migratory capacities have been likened to leukocyte movement toward sites of inflammation; some possible pathways and mechanisms have been discussed in recent reviews.97,152 Homing of MSCs is more complex and not yet as clearly understood as their tumor tropism. Homing capacity of MSCs can be defined as the cell’s tendency to return to their site of extraction. Several reports indicated that variations in protocols used for isolation and expansion of MSCs ex vivo affects their homing capacity. Kemp et al.153 suggested that both the phenotype and homing capacity of MSCs can be altered by the duration of passaging time.153 Furthermore, progressive passaging of the cells is associated with a decrease or loss of chemokine receptors (i.e. CXCR4), adhesion molecule expression, and lack of further chemotactic response.146,154 Moreover, the confluency rate has been shown by De Becker et al.155 to be directly proportional to the production of the tissue inhibitor of metalloproteinase-3 (TIMP-3) and inhibiting trans-endothelial migration.155 Although there is no question with regards to the migration and integration capacity of MSCs towards tumor tissue, their exact function inside the tumor remains ambiguous. With a better understanding of MSC-tumor interactions, one can deduce more thorough conclusions in regards to the role they have in tumor development or destruction. Next we aim to address both the protumorigenic and antitumorigenic properties of MSCs. Pro-oncogenesis properties of MSCs The anatomy of a tumor consists mainly of two separate but interlinked compartments: the parenchyma and the stroma. The parenchyma compartment is better known as the neoplastic compartment (malignant cells) and the stroma is the nonmalignant supportive tissues that includes: extracellular matrix & 2014 Nature America, Inc.

(ECM), blood vessels, immune and inflammatory cells, connective tissues, and MSCs.156 The stroma has a critical role in tumor growth as it acts as an intermediate structure between the tumor microenvironment and host tissues. It is believed that change in either of the two compartments could contribute to accelerated tumor growth. Studies indicating the protumorigenic capacity of MSCs have shown that MSC-induced immunosuppression capabilities, MSC-mediated angiogenesis, epithelial–mesenchymal transition (EMT)-mediated enhancement of tumor stroma and potential malignant transformation could have a vital role in tumor growth and formation. IMMUNOSUPPRESSION Influence of MSCs on both the innate and cellular immune pathways has been widely observed in vitro. Almand et al.157 have shown that in peripheral blood obtained from cancer patients, a higher frequency of MSCs with suppressive functions was observed.157 For MSCs to be incorporated into a tumor, a capacity to downregulate the immune system is necessary.158,159 Interferon-gamma alone or with the aid of TNF-a, IL-1a, or IL-1b is thought to activate MSCs.160 This MSC activation is observed in the interferon-gamma knockout mouse model of graft-versus-host disease and shows that in the absence of IFN receptors, MSCs will not become activated.161 In addition, inhibition of lymphocyte proliferation and suppression of the immune function of effector T lymphocytes (Teff), such as: CD4 þ and CD8 þ T cells, B cells, and natural killer cells have been observed by MSCs.131,162 The interaction and influence of MSCs on the immune system has proven to be more complex than previously thought, and it appears that this could be attributed to direct cell interactions between MSCs and other soluble factors that are released from MSCs. Lymphocyte inhibition The molecular mechanisms involved in the inhibition of T lymphocytes via MSCs are not fully understood. Different mechanisms have been proposed such as cell cycle arrest,163,164 immunomodulatory activity of growth factors and other molecules secreted by MSCs,165–167 and influence of the maturation of dendritic cells.167 Regulatory T cells (Treg) The role of Tregs in the immune system response has been studied extensively. It is widely accepted that Tregs are capable of downregulating proliferation of other T cells through a cell–cell contact mechanism, IL10, and TGF-b secretion. Recent in vitro experiments demonstrate induced generation of Tregs via human MSCs.139,167,168 This differentiation of Tregs can be explained by the release of TGF-b and sHLA-G5 by MSCs. Patel et al.139 compared levels of Treg populations before and after co-cultures of breast cancer cells with MSCs and observed a nearly twofold increase of Treg in the co-cultures compared with controls.139 They further concluded that TGF-b1 secreted by MSCs is largely responsible for this increase in population. Research indicates the importance of Tregs in the microenvironment of human tumors. A subset of CD4 þ CD25high Foxp3 þ cells (Tregs) are expressed more significantly in tumor microenvironments than the frequency observed in the peripheral circulation of patients with cancer.169–171 To this end, more follow-up studies of MSC-induced Tregs function are still needed to fully understand the mechanism of how MSCs function to regulate Tregs. MSC-MEDIATED ANGIOGENESIS Optimal tumor growth is highly dependent on the angiogenesis capacity of the tumor site that could have a profound impact in metastasis of the tumor itself. Frequently, tumor necrosis is seen in Cancer Gene Therapy (2014), 12 – 23


18 the inner layers of solid tumors where vascularization occurs at a much lower density due to an inability to match the speed of the tumor cell growth. Thus, any therapy involved with antiangiogenesis is considered of vital importance in the field of cancer therapy. Activation of endothelial cells has been reported through soluble factors and MSC’s cell-to-cell contacts.172 The expression of proangiogenic factors such as angiopoietins-1, vascular endothelial growth factor, platelet-derived growth factor, and fibroblast growth factors, FGF-2 and FGF-7 have been observed in MSCs.154,173 The expression of receptors such as IL-8 on the MSCs encourages recruitment of endothelial progenitors154 and these factors are known to induce angiogenesis and rapid vascularization. Chopp and Li 127 demonstrated that MSCs or their supernatant derived from culture induce significant angiogenesis in cornea treatments.127 They further concluded that the supernatant is even more effective than direct use of growth factors such as vascular endothelial growth factor. Generation of vessel-like tubular structures has been observed upon transplantation of MSCs in Matrigel plugs in vivo.174 Overall, the role of MSCs in angiogenesis in a tumor microenvironment still remains very complex and more in-depth studies are needed to reach definitive conclusions. EPITHELIAL–MESENCHYMAL TRANSITION EMT is best characterized as a stage in which cells are modified in their adhesion capabilities, polarity, cytoskeletal system, expression levels of intermediate filaments, motility, and resistance to anoikis.175,176 EMT is associated with the epithelial-derived cancers, representing nearly 90% of human cancers.176 This characteristic is especially important during organogenesis of the embryonic stage and wound healing. It can be deduced that if EMT occurs in an uncontrolled manner it could facilitate the progression and metastasis of cancer. Iwatsuki et al.177 describe that cancer cells undergoing this transition become more aggressive and progressive in nature leading to metastasis.177 In a recent study on breast cancer, Martin et al.178 verified that significant upregulation of EMT occurs upon co-culture of MSCs with cancer cells.178 The observed changes were due to cell contact mediation and via MSCs. POTENTIAL MALIGNANCY OF MSCS Fear of malignant transformation of stem cells has been a significant concern in cell-based therapies. Similarly, the potential of malignant transformation of MSCs has been a major concern. Human MSCs were identified to differ from their murine counterparts, as they did not go through spontaneous transformation.179 Berrnardo et al.180 attribute this finding to the loss of replicatively senescent human MSCs during long-term culture, and conclude that using human MSCs are therapeutically safe.180 Alternatively, other studies done by Wang et al.181 and Rubio et al.182 have shown opposite findings.181,182 Houghton et al.183 reported that bone marrow-derived MSCs could lead to gastric cancer.183 Although there are different findings associated with the malignant transformation of the MSCs, there is a consensus to avoid unnecessary manipulation and prolonged passaging for MSCs used for therapy.128,131 Next, we will consider some of the mechanisms by which MSCs possess anticancer effect on tumor tissue. Anti-oncogenesis effects of MSCs The antitumorigenic properties of MSCs have been attracting immense scientific attention while different animal models have indicated the inhibition of tumor growth via MSCs. Maestroni et al.184 initially reported inhibition of tumor growth and metastasis by co-injection of murine MSCs and tumor cells in Cancer Gene Therapy (2014), 12 – 23

lung cancer and melanoma mice models.184 By using a subcutaneous co-transplantation model of colon cancer cells and MSCs in rats, Ohlsson et al.185 demonstrated the tumor inhibition capabilities of MSCs.185 Recently, studies have shown the intrinsic capability of MSCs to produce antitumorigenic effects in pancreatic cancer, hepatic cancer, and Kaposi’s sarcoma.141,186,187 Next, we will describe three possible mechanisms by which MSCs impose their antitumor effects: downregulation of Wnt and Akt pathways, and the intrinsic antitumor capability of MSCs. DOWNREGULATION OF WNT SIGNALING PATHWAY Although cancer and stem cells have very unique and different origins, they share many biological characteristics.188 Many signaling pathways that regulate differentiation, cell survival, and cell death including Wnt, Notch, Shh, and BMP are shared by both cell types.189–191 The Wnt pathway is extensively involved in stem cell self-renewal and differentiation. While abnormalities in this pathway have been related to human tumor progression and expansion;192,193 this pathway also has a critical role in pancreatic cancer development as discussed in section ‘Wnt/-catenin Pathway and Pancreatic Cancer’. In a study done by Qiao et al.187 MSC-mediated downregulation of Wnt signaling pathway was observed using a hepatoma animal model.187 Through their findings, they showed that during co-injection of human hepatoma cells (H7420) and human MSCs, the rate of proliferation in H7420 cells decreased dramatically, with an increased rate of apoptosis and downregulation of all targets of the Wnt pathway (Bcl-2, cMyc, PCNA, and survivin) most likely via producing inhibitors of Wnt. In addition, they concluded that this observation is correlated with the paracrine function of MSCs as when they used the conditioned media, similar results were observed. Further studies are required to understand the mechanisms of Wnt pathway-induced apoptosis via MSCs in tumor microenvironment. DOWNREGULATION OF AKT PATHWAY Opposite to what was observed in the Wnt signaling pathway study in hepatoma models, the MSC-mediated downregulation of Akt pathways required direct cell contact in a Kaposi’s sarcoma model. As discussed in section ‘PI3K/AKT/mTOR pathway and pancreatic cancer, amplification of the Akt pathway is a repeated theme in pancreatic as well as other cancer models. This molecule has been the hallmark of human Kaposi’s sarcoma. Khakoo et al.141 demonstrated that intravenous injection of MSCs resulted in their homing to the site of the tumor as early as 48 h that effectively inhibited the tumor growth.141 In this in vitro study, an inhibition of the activation of Akt protein kinase via MSCs was observed. They demonstrated that for this inhibition to occur, direct cell contact is required and they were able to reverse the inhibition by anti-antibody against E-cadherin molecules.141 In their in vivo model, inhibition of Akt activation was observed in areas neighboring MSC infiltration.141 These findings confirm the antitumorigenic properties of MSCs via the Akt pathway and also showed that deregulated Akt may be a specific target of MSCs in the tumor microenvironment. INTRINSIC ANTITUMOR PROPERTIES The intrinsic antitumor properties of MSCs have been observed in a number of studies. Lu et al.194 displayed cell cycle arrest in response to MSCs in lymphoma, hepatoma, and insulinoma cells, which was followed with an increased rate of apoptosis in the respective tumor cells and, reduction in malignant ascites in vivo and in vitro.194 This observation has also been seen in other cancer models using MSCs. In pancreatic cancer, the intraperitoneal injection of MSCs reduced the tumor growth rate and improved & 2014 Nature America, Inc.


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Figure 3. Expected personalized treatment of pancreatic cancer with engineered mesenchymal stem cells (MSCs:). (a) Displays the proposed effect of using engineered MSCs from bedside to bench side and vice versa. Patients MSCs are extracted and expanded in vitro they are then engineered with desired antitumor genes and are tested against patient’s cancer cells. Once the best candidate genes are identified they will be mass engineered and transplant into the patients. We anticipate that with MSC’s unique properties, we would get a much more specific treatment option. (b) is an schematic of conventional use of chemotherapy drugs for the treatment of pancreatic cancer and its effect on all body parts. Worth mentioning is the lack of personalized approach in the current conventional therapies.

longevity in a SCID mice model.186 In a breast cancer model, both the intratumoral and IV injections of naive MSCs lead to reduced tumor growth and increased cancer cell apoptosis.195 The actual mechanisms stimulating these intrinsic antitumor properties in MSCs remains complex and further research is required to discern what influences are activating these results, while downregulation of Akt, Wnt, and NFkB pathways have been reported to be of great importance in the explanation of these intrinsic MSC properties.196 Figure 2 summarizes both the protumorigenic and antitumorigenic effects of MSCs in a tumor microenvironment. Results of such investigations may vary due to influences of primary tumor origin, location and subtype, as well as the MSC isolation protocol, time of propagation and the route of administration. It is evident that conflicting data in regards to the role of MSCs in tumor microenvironments indicate that this field of research is still in its early phases and more conclusive work is necessary to substantiate and solidify our knowledge in this area.

MSCs have an immense potential to be used in either the auto- or allotransplantation discipline of cancer or regenerative medicine. Our goal remains to expand on the antitumor properties of MSCs while incorporating their other potential benefits and developing them as a vehicle for delivery of anticancer genes to cancer targets. MSCs engineered with anticancer genes are capable of specifically attacking tumor cells through multiple mechanisms: (1) tumor-directed migration and incorporation; (2) anticancer agent delivery, and (3) organ-specific vector construction. The work in this study focuses on the first two of these mechanisms. With the extensive variations that exist amongst patients, we aim to use multiple anticancer genes in an effort to determine which provides a more customized approach to yielding maximal killing of the targeted cancer site. For translation of this research into clinical settings, personalized therapy for each patient is critical. This is summarized in Figure 3 that illustrates a general scheme of putative personalized treatment with anticancer gene-engineered MSCs.

The potential personalized medicine of engineered MSCs As science advances and knowledge expands about how each patient is unique in their individual reaction to a therapy, more attention is transferred to the theme of personalized medicine.

SUMMARY AND CONCLUSION Pancreatic cancer remains one of the most challenging cancer types to treat. Current conventional therapies for pancreatic cancer are passive and symptomatic in nature. With the pressing


Cancer Gene Therapy (2014), 12 – 23


20 demand on scientists to find new breakthrough therapeutic strategies, using MSCs combined with anticancer agents hold great promise. MSCs can be obtained from many different organs. One source that is widely studied in the field has been the bone marrow-derived MSCs. Although the characteristics between MSCs across other organs are very similar, the individual mechanistic differences between MSCs derived from different organs in in vivo or in vitro models remains a mystery. Properties of bone marrowderived versus pancreatic-derived MSCs could be customdesigned to exert maximal impact on treatments for pancreas diseases. Although previous investigations have shown the homing capacity of bone marrow-derived MSCs in different animal models, the promise of pancreatic-derived MSCs is that they could show a stronger homing capacity to target neoplasms of pancreatic tissue origin. This study provides a practical platform for the development of MSC-based and personalized treatment of pancreatic cancer.

CONFLICT OF INTEREST The authors declare no conflict of interest.

ACKNOWLEDGEMENTS The work was supported by the VGH and UBC Hospital Foundation. We are grateful for Mrs Crystal Robertson’s assistance for manuscript editing.

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