Stromal Targeting Research Articles

Radiation-enhanced therapeutic targeting of galectin-1 enriched malignant stroma in triple negative breast cancer.

Currently there are no FDA approved targeted therapies for Triple Negative Breast Cancer (TNBC). Ongoing clinical trials for TNBC have focused primarily on targeting the epithelial cancer cells. However, targeted delivery of cytotoxic payloads to the non-transformed tumor associated-endothelium can prove to be an alternate approach that is currently unexplored. The present study is supported by recent findings on elevated expression of stromal galectin-1 in clinical samples of TNBC and our ongoing findings on stromal targeting of radiation induced galectin-1 by the anginex-conjugated arsenic-cisplatin loaded liposomes using a novel murine tumor model. We demonstrate inhibition of tumor growth and metastasis in response to the multimodal nanotherapeutic strategy using a TNBC model with orthotopic tumors originating from 3D tumor tissue analogs (TTA) comprised of tumor cells, endothelial cells and fibroblasts. The ‘rigorous’ combined treatment regimen of radiation and targeted liposomes is also shown to be well tolerated. More importantly, the results presented provide a means to exploit clinically relevant radiation dose for concurrent receptor mediated enhanced delivery of chemotherapy while limiting overall toxicity. The proposed study is significant as it falls in line with developing combinatorial therapeutic approaches for stroma-directed tumor targeting using tumor models that have an appropriate representation of the TNBC microenvironment.

Editorial: Novel Targets and Targeting Technologies to Modulate Tumor Microenvironment.

Editorial: Novel Targets and Targeting Technologies to Modulate Tumor Microenvironment.

Stromal Targets for Fluorescent-Guided Oncologic Surgery.

Pre-operative imaging techniques are essential for tumor detection and diagnosis, but offer limited help during surgery. Recently, the applicability of imaging during oncologic surgery has been recognized, using near-infrared fluorescent dyes conjugated to targeting antibodies, peptides, or other vehicles. Image-guided oncologic surgery (IGOS) assists the surgeFon to distinguish tumor from normal tissue during operation, and can aid in recognizing vital structures. IGOS relies on an optimized combination of a dedicated fluorescent camera system and specific probes for targeting. IGOS probes for clinical use are not widely available yet, but numerous pre-clinical studies have been published and clinical trials are being established or prepared. Most of the investigated probes are based on antibodies or peptides against proteins on the membranes of malignant cells, whereas others are directed against stromal cells. Targeting stroma cells for IGOS has several advantages. Besides the high stromal content in more aggressive tumor types, the stroma is often primarily located at the periphery/invasive front of the tumor, which makes stromal targets particularly suited for imaging purposes. Moreover, because stroma up-regulation is a physiological reaction, most proteins to be targeted on these cells are “universal” and not derived from a specific genetic variation, as is the case with many upregulated proteins on malignant cancer cells.

A tenascin C targeted nanoliposome with navitoclax for specifically eradicating of cancer-associated fibroblasts

Cancer-associated fibroblasts (CAFs) play a vitally important role during tumor progression. Navitoclax (Nav) can specifically induce apoptosis in CAFs. The present study aims to develop a novel CAF-targeted nanoliposome for cancer therapy. Nav-loaded nanoliposomes modified with peptide FH (FH-SSL-Nav), which specifically binds to tenascin C, a protein mainly expressed by CAFs, were formulated and characterized. Several experiments were performed to evaluate CAFs selective apoptosis, targeting and eradicating. Compared with SSL-Nav, FH-SSL-Nav achieved higher cellular uptake, and exhibited stronger cytotoxicity in vitro. The in vivo tumor stroma targeting effect was further confirmed by near infrared imaging. Accordingly, FH-SSL-Nav displayed improved tumor growth inhibition by eradicating CAFs in Hep G2 tumor-bearing nude mice model. In conclusion, FH-SSL-Nav could achieve targeting delivery of Nav to CAFs in vitro and in vivo, and may offer a potential strategy for cancer therapy based on tumor stroma. From the Clinical EditorThe progression of cancer cells often depends on the underlying tumor microenvironment, in which cancer-associated fibroblasts play an important role. In this article, the authors developed targeted therapy against CAFs using liposomes as carriers. This new modality was shown to be more effective in tumor killing both in vitro and in vivo. The finding may open a new era in cancer therapy.

Targeting Artificial Tumor Stromal Targets for Molecular Imaging of Tumor Vascular Hypoxia.

Developed and tested for many years, a variety of tumor hypoxia detection methods have been inconsistent in their ability to predict treatment outcomes or monitor treatment efficacy, limiting their present prognostic capability. These variable results might stem from the fact that these approaches are based on inherently wide-ranging global tumor oxygenation levels based on uncertain influences of necrotic regions present in most solid tumors. Here, we have developed a novel non-invasive and specific method for tumor vessel hypoxia detection, as hypoxemia (vascular hypoxia) has been implicated as a key driver of malignant progression, therapy resistance and metastasis. This method is based on high-frequency ultrasound imaging of α-pimonidazole targeted-microbubbles to the exogenously administered hypoxia marker pimonidazole. The degree of tumor vessel hypoxia was assessed in three mouse models of mammary gland carcinoma (4T1, SCK and MMTV-Wnt-1) and amassed up to 20% of the tumor vasculature. In the 4T1 mammary gland carcinoma model, the signal strength of α-pimonidazole targeted-microbubbles was on average 8-fold fold higher in tumors of pimonidazole-injected mice than in non-pimonidazole injected tumor bearing mice or non-targeted microbubbles in pimonidazole-injected tumor bearing mice. Overall, this provides proof of principle for generating and targeting artificial antigens able to be ‘created’ on-demand under tumor specific microenvironmental conditions, providing translational diagnostic, therapeutic and treatment planning potential in cancer and other hypoxia-associated diseases or conditions.

Revisiting stroma in pancreatic cancer.

Revisiting stroma in pancreatic cancer.

Abstract 2389: Eps8: a negative regulator of myofibroblast differentiation and function

Abstract In this study we investigate the mechanisms regulating myofibroblast differentiation and function, and identify the adapter protein Epidermal growth factor receptor kinase substrate 8 (Eps8) as a negative regulator of the cancer-associated myofibroblast phenotype. Cancer-associated fibroblasts (CAFs) are a heterogenous, poorly defined cell population. Most commonly CAFs are identified by de novo expression of α-smooth muscle actin (αSMA) incorporated into stress fibres generating enhanced contractility, which is associated with an enhanced secretory profile. Myofibroblasts promote a number of the hallmarks of malignancy, and clinical studies have shown that the presence of an αSMA-rich stroma is associated with poor outcome in several cancers, suggesting that stromal targeting may be a useful therapeutic adjunct. The aim of this study was to investigate the mechanisms regulating differentiation of the myofibroblastic CAF phenotype. Fibroblasts (HFFF2 and primary fibroblasts extracted and cultured from normal skin, esophageal and oral mucosa) were treated with TGF-β1 to induce myofibroblast transdifferentiation. Target genes were suppressed using siRNA. Gene expression was examined using RNA-seq and qRT-PCR, and protein expression by Western blotting and immunofluorescence microscopy. Myofibroblast function was examined using gel contraction assays while the effect of the myofibroblast secretion profile on cancer cell migration was assessed using Transwell® migration assays. RNA-seq confirmed the upregulation of myofibroblast-associated genes (FN1, COL1A1, CTGF, ACTA2) in TGF-β1-treated fibroblasts. Conversely, we found that the adaptor protein Eps8 was significantly down-regulated, and this occurred early in the transdifferentiation process. Eps8 sits at the heart of a complex system regulating reorganization of the actin cytoskeleton, and interacts with numerous intracellular proteins including palladin, F-actin and integrins. We found that siRNA knockdown of Eps8 in fibroblasts promoted myofibroblast differentiation, resulting in increased expression of αSMA, stress fibre formation and cell contractility and promotion of cancer cell motility. Suppression of Eps8 expression sensitized fibroblasts to TGF-β1 treatment and potentiated TGF-β1 signalling, in part through transcriptional upregulation and increased phosphorylation of SMAD2. These data suggest that Eps8 expression provides tonic inhibition of myofibroblast transdifferentiation, at least in part by desensitization of cells to TGF-β1 mediated by the regulation of SMAD2 expression. Loss of Eps8 expression may play an important role in the generation of an αSMA-positive myofibroblastic CAF population. Citation Format: Steven J. Frampton, Veronika Jenei, Massimiliano Mellone, Christopher J. Hanley, Marta M. Rucka, Joanne Tod, Karwan A. Moutasim, Emma V. King, Gareth Thomas. Eps8: a negative regulator of myofibroblast differentiation and function. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2389. doi:10.1158/1538-7445.AM2015-2389

Highlights of This Issue

Whatcott and colleagues examined extracellular matrix deposition in clinical samples of both primary pancreatic tumors and metastases from various sites. Looking at collagen I levels, they observed that primary tumors and metastatic lesions show comparable levels of desmoplasia. They also report a negative correlation between patient survival and extracellular matrix (Collagen I or Hyaluronan) deposition. As stromal targeting is emerging as an alternative approach to enhance chemotherapeutic interventions, their analysis provides further support for use of the approach, even in patients with late stage, metastatic disease.In follicular lymphoma, the tumor microenvironment has been shown to influence patient outcomes. To clarify the association of tumor-associated macrophages (TAMs) with prognosis, Kridel and colleagues performed immunohistochemistry for CD163 in two large patient cohorts. In cases from British Columbia who had been treated with rituximab, cyclophosphamide, vincristine, and prednisone (R-CVP), CD163+, macrophages were associated with poor prognosis, but an opposite outcome correlation was observed in those patients from the PRIMA trial who received an anthracycline in addition to R-CVP (R-CHOP). These data show that the prognostic impact of TAMs is highly dependent on patient characteristics and/or treatment received.Glycodelin, an immunomodulatory protein, is highly expressed during the menstruation cycle and pregnancy. In the current study, Schneider and colleagues demonstrated a high expression of glycodelin in NSCLC. A knockdown of glycodelin mRNA resulted in a deregulation of immune system modulatory proteins, such as PDL1/PDL2 and MICA/MICB, and a reduced migration of NSCLC cell lines. Glycodelin was secreted from NSCLC tumors and could be measured in patients' serum. Furthermore, the serum levels correlated with the tumor response to therapy and therefore demonstrated that glycodelin can be used as a helpful follow-up biomarker.The prevalence of a pretreatment EGFR T790M mutation in patients with EGFR-mutant non-small cell lung cancer (NSCLC) remains unclear. To assess this question, and its clinical significance, Watanabe and colleagues used an ultra-sensitive droplet digital PCR technique. Surgically resected tumor tissues from 373 early-stage NSCLC patients with EGFR-activating mutations were examined, and the overall incidence of the pretreatment EGFR T790M mutation was 79.9% (298/373). The mutant allele frequency ranged from 0.009% to 26.9%. These data suggest that an ultra-sensitive method can provide high resolution mutational profiling, and open up the possibility of developing new treatment strategies with EGFR-TKIs, including third-generation TKIs for patients with EGFR-mutant NSCLC.

Abstract 3545: Stromal selective targeting by uPAR retargeted oncolytic measles virus inhibits breast cancer progression

Abstract Stromal cells in the tumor microenvironment play an important role in breast cancer progression. Strategies directed against stromal cell components have been investigated in the preclinical and clinical setting. However, few studies have focused on stromal targeting by oncolytic viruses. The urokinase receptor (uPAR) is overexpressed in tumor and stromal cells, and plays a critical role in tumor progression. The aim of this study is to evaluate the antitumor effects of stromal retargeted oncolytic measles virus via urokinase receptor. We rescued and characterized fully retargeted oncolytic measles viruses against human (MV-h-uPA) and murine (MV-m-uPA) urokinase receptor (uPAR). In vitro, MV-m-uPA and MV-h-uPA infected and induced cytotoxicity to human (MDA-MB231, T47D and MCF-7) and murine (4T1) mammary cancer cells in a species specific manner. Efficient MV-m-uPA infection and replication were demonstrated in murine NIH-3T3 mouse fibroblast cells and MS1 murine endothelial cells. Selective infection of murine fibroblasts by the murine uPAR specific MV-m-uPA led to significant inhibition of human breast cancer proliferation in an in vitro 3-D collagen co-culture model of tumor-stromal interactions. To further validate the potential of stromal uPAR targeting as a therapeutic strategy, the effects of MV-uPA were assessed in a human breast cancer xenograft model (MDA-MD231), where tumor cells express human uPAR (target of MV-h-uPA) and the host stroma expresses murine uPAR (target of MV-m-uPA). Tumor bearing mice were treated with MV-h-uPA, MV-m-uPA or combination (MV-h-uPA and MV-m-uPA) by IV administration (3 doses). Surprisingly, MV-m-uPA (selectively targeting mouse uPAR) was associated with a measurable delay in tumor progression while on treatment, as well as prolongation in survival. Combination therapy (MV-m-uPA and MV-h-uPA) was associated with the best antitumor effects in vivo. To further characterize the in vivo effects of stromal uPAR targeting, repeat experiments were performed where tumor bearing mice received prolonged treatment with MV-m-uPA (12 treatments). This approach was shown to be safe and resulted in a significant delay in tumor progression and marked prolongation of survival. These effects were associated with the identification of viable viral particles and viral RNA from MDA-MB231 tumor xenograft treated with MV-m-uPA. In conclusion, our results demonstrate for the first time that oncolytic viral targeting of tumor stroma is feasible and associated with in vivo antitumor effects. These findings further validate the critical role of stromal uPAR in cancer progression. Citation Format: Yuqi Jing, Marcela Toro Bejarano, Krisztina Kovacs, Jaime Merchan. Stromal selective targeting by uPAR retargeted oncolytic measles virus inhibits breast cancer progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3545. doi:10.1158/1538-7445.AM2015-3545

PTU-126 Stromal targeting with phosphodiesterase type 5 inhibitors in oesophageal adenocarcinoma

Introduction The UK has the highest age-standardised incidence of oesophageal adenocarcinoma (EAC) worldwide. Deaths from EAC have risen by 50% since the 1970’s and 10-year survival is 1 in 10. An activated stroma (defined by α-SMA positive, Cancer Associated Fibroblasts (CAF)) is associated with poor prognosis across tumour types. We have shown the presence of CAF to be more predictive of survival in EAC than traditional pathological markers. 1 Stromal targeting is a novel area in cancer treatment, no currently licensed drugs are effective in targeting CAF. We describe the repurposing of Phosphodiesterase Type 5 inhibitors (PDE5i) for use in the treatment of EAC. Method The effect of PDE5i on EAC CAF was studied using small molecule inhibitors and siRNA knock down in primary cell culture and functional assays; collagen gel contraction, Transwell® invasion and 3D organotypic culture. Weighted correlation gene network analysis (WCGNA) was carried out on publically available gene microarray. Results PDE5 expression was upregulated in CAF extracted from EAC patients, compared to matched normal fibroblasts (n = 3). PDE5i significantly decreased the expression of α-SMA by CAF. Functional effects were investigated and a significant decrease in collagen-1 gel contraction (p = 0.012) was observed. Cancer cell migration (p Conclusion Stromal targeting by CAF inactivation is a new potential treatment for hard to treat solid organ malignancies. We have compelling evidence that PDE5i treatment of EAC CAF significantly decreases; α-SMA expression, contractility and the ability to promote cancer cell invasion. Recent evidence supports the role of CAFs in tumourigenesis and immune modulation, 2,3 PDE5 inhibition of the CAF phenotpye offers the possibility of increased drug deliver (chemo/immunotherapy) and host immune cell infiltration, decreased local and distant cancer growth and invasion. The time is right for the first human trial of PDE5i as an anti-cancer therapy. Disclosure of interest None Declared. References Underwood et al . Cancer-associated fibroblasts predict poor outcome and promote periostin-dependent invasion in oesophageal adenocarcinoma. J Pathol. 2015 Han et al . Molecular mechanism underlying the tumor-promoting functions of carcinoma-associated fibroblasts. Tumour Biol. 2015 Fearon, et al . The carcinoma-associated fibroblast expressing fibroblast activation protein and escape from immune surveillance. Cancer Immunol Res . 2014

EBV oncogene N-LMP1 induces CD4 T cell-mediated angiogenic blockade in the murine tumor model.

Antivascular immunity may provide long-term protection by preventing neovascularization that precedes tumor progression. Although the tumorigenesis promoted by EBV-encoded oncogene latent membrane protein 1 derived from Taiwanese nasopharyngeal carcinoma (N-LMP1) has been demonstrated, the potential of N-LMP1 for inducing immune surveillance remains elusive. In this article, we describe the immunogenicity of N-LMP1 (1510) and its induction of antivascular immunity in a transplantable tumor model in immunocompetent BALB/c mice. The immunogenicity of N-LMP1 was evaluated on the basis of tumor rejection following immunization. The impact of the immunization on the dynamics of tumor angiogenesis was assessed by temporal noninvasive dynamic contrast-enhanced magnetic resonance imaging and was further confirmed by histologic study and vascular count. Through the experiments of in vivo depletion and adoptive transfer, CD4 T cells were identified as effectors that depend on IFN-γ for tumor prevention. The response was further verified by the identification of an MHC H-2 I-E(d)-restricted peptide derived from N-LMP1 and by the immunization of mice with N-LMP1 peptide-loaded dendritic cells. These studies provide insight into N-LMP1-specific immunity in vivo, which suggests that CD4 T cells may play an important role in angiogenic surveillance against LMP1-associated cancer via tumor stroma targeting.

Sa1687 Modeling Cholangiocarcinoma Desmoplasia for Rapidly Identifying Stromal Targeting Agents

A S L D A b st ra ct s sham or bile duct ligation (BDL) and were then treated with chloroquine (an inhibitor of autophagy; 60 mg/kg/day), rapamycin (an activator of autophagy; 30 mg/kg/day) or recombinant PGRN (5 nmol/kg/day) via intraperitoneal implanted minipump for 3 days. Cholangiocyte proliferation and intrahepatic biliary mass were assessed in vivo by immunohistochemistry using proliferating cellular nuclear antigen (PCNA) or cytokeratin 19 (CK19) antibodies respectively. In vitro, a parental mouse cholangiocyte cell line and cells overexpressing the pro-autophagic effector Sirt1 were treated with chloroquin and rapamycin and PGRN for up to 48 hr and proliferation assessed using MTS assays. Expression of the autophagy markers, Beclin, ATG5, ATG7 and LAMP1, and Sirt1 were assessed by qPCR, immunoblotting and immunohistochemistry in livers and cholangiocytes after the above-mentioned treatments. Autophagy was also assessed in vitro in mouse cholangiocyte cell lines using a commercially available lysotracker staining kit. Results: After BDL, markers of autophagy were increased, specifically in cholangiocytes. Strategies to inhibit autophagy (chloroquine) increased intrahepatic biliary mass and induced cholangiocyte proliferation in vivo and in vitro, whereas strategies to stimulate autophagy (rapamycin) reduced intrahepatic biliary mass after BDL and reduced cholangiocyte proliferation in vivo and in vitro. Treatment with recombinant PGRN reduced the expression of autophagy markers in cholangiocytes and increased cholangiocyte proliferation. Associated with these effects was a PGRN-induced suppression of the pro-autophagic molecule, Sirt1. Indeed, the inhibitory effect of PGRN on autophagy was alleviated in cholangiocytes overexpressing Sirt1. Conclusion: Our data suggest that autophagy is upregulated during hyperplastic cholangiocyte proliferation, presumably as a compensatory mechanism to prevent overt proliferation. Furthermore, PGRN may induce cholangiocyte proliferation by inhibiting autophagy via the suppression of Sirt1 expression.

The conflicting roles of tumor stroma in pancreatic cancer and their contribution to the failure of clinical trials: a systematic review and critical appraisal

A nearly universal feature of pancreatic ductal adenocarcinoma (PDAC) is an extensive presence of activated stroma. This stroma is thought to aid in various tumor-promoting processes and hampers response to therapy. Here, we aim to evaluate the evidence that supports this role of the stroma in PDAC with functional experiments in relevant models, discuss the clinical trials that have aimed to target the stroma in this disease, and examine recent work that explains why these clinical trials based on stroma-targeting strategies have thus far not achieved the expected success. We systematically searched PubMed through August 2014 with no restrictions to identify published peer-reviewed research articles assessing the effect of targeting the stroma on tumor growth or metastases in preclinical animal models. Five hundred and thirty articles were extracted of which 31 were included in the analysis. Unfortunately, due to the large variety in models and outcome measures, we could not perform a meta-analysis of our data. We find that despite an abundance of positive outcomes reported in previous studies on stroma targeting, a strong discrepancy exists with the outcomes of clinical trials and the more recent preclinical work that is in line with these trials. We explain the incongruities by the duration of stroma targeting and propose that chronic stroma targeting treatment is possibly detrimental in the treatment of this disease.

Targeting Artificial Tumor Stromal Targets for Molecular Imaging of Tumor Vascular Hypoxia

Targeting Artificial Tumor Stromal Targets for Molecular Imaging of Tumor Vascular Hypoxia

Efficacy of CAR T-cell therapy in large tumors relies upon stromal targeting by IFNγ.

Adoptive T-cell therapy using chimeric antigen receptor-modified T cells (CAR-T therapy) has shown dramatic efficacy in patients with circulating lymphoma. However, eradication of solid tumors with CAR-T therapy has not been reported yet to be efficacious. In solid tumors, stroma destruction, due to MHC-restricted cross-presentation of tumor antigens to T cells, may be essential. However, CAR-Ts recognize antigens in an MHC-independent manner on cancer cells but not stroma cells. In this report, we show how CAR-Ts can be engineered to eradicate large established tumors with provision of a suitable CD28 costimulatory signal. In an HER2-dependent tumor model, tumor rejection by HER2-specific CAR-Ts was associated with sustained influx and proliferation of the adoptively transferred T cells. Interestingly, tumor rejection did not involve natural killer cells but was associated instead with a marked increase in the level of M1 macrophages and a requirement for IFNγ receptor expression on tumor stroma cells. Our results argue that CAR-T therapy is capable of eradicating solid tumors through a combination of antigen-independent stroma destruction and antigen-specific tumor cell targeting.

Image-based RNA interference screening reveals an individual dependence of acute lymphoblastic leukemia on stromal cysteine support.

Interactions with the bone marrow microenvironment are essential for leukemia survival and disease progression. We developed an imaging-based RNAi platform to identify protective cues from bone marrow derived mesenchymal stromal cells (MSC) that promote survival of primary acute lymphoblastic leukemia (ALL) cells. Using a candidate gene approach, we detected distinct responses of individual ALL cases to RNA interference with stromal targets. The strongest effects were observed when interfering with solute carrier family 3 member 2 (SLC3A2) expression, which forms the cystine transporter xc- when associated with SLC7A11. Import of cystine and metabolism to cysteine by stromal cells provides the limiting substrate to generate and maintain glutathione in ALL. This metabolic interaction reduces oxidative stress in ALL cells that depend on stromal xc-. Indeed, cysteine depletion using cysteine dioxygenase resulted in leukemia cell death. Thus, functional evaluation of intercellular interactions between leukemia cells and their microenvironment identifies a selective dependency of ALL cells on stromal metabolism for a relevant subgroup of cases, providing new opportunities to develop more personalized approaches to leukemia treatment.

Abstract 191: Desmoplasia in primary tumors and metastatic lesions of pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a major cause of cancer-related death in the United States. PDAC is characterized by high levels of fibrosis, a feature termed desmoplasia. Desmoplasia is thought to hamper the efficacy of therapeutics treating PDAC. Agents targeting components of the tumor stroma have shown some enhancement of first-line therapeutics targeting tumor cells. As metastatic burden is one of the primary causes for mortality in PDAC, the question remains of whether stromal targeting therapeutics will benefit patients with late stage, metastatic disease. We sought to address this question by assessing the extent of desmoplasia in both primary PDAC tumors and metastatic lesions of pancreatic origin. Patient tissue staining using a pentachrome or standard immunohistochemical technique demonstrates that extracellular matrix components, such as collagen, are found in high levels in both primary tumors and metastatic lesions. The difference in the level of desmoplasia in primary tumors and metastatic lesions was statistically insignificant. We also observed a significant correlation between patient survival and extracellular matrix deposition. Kaplan-meier curves for collagen I showed median survival of 527 days in low collagen patients, and 176.5 in high level patients. Low level hyaluronan patients displayed median survival times of 2107 days as compared to 385 days in high level patients. Our results indicate that both primary tumors and metastases of PDAC have highly fibrotic stroma. Thus, stromal targeting agents may benefit PDAC patients, even those with metastatic disease. Citation Format: Clifford J. Whatcott, Aprill Watanabe, Janine LoBello, Daniel Von Hoff, Haiyong Han. Desmoplasia in primary tumors and metastatic lesions of pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 191. doi:10.1158/1538-7445.AM2014-191

Abstract 4799: Identification and characterization of stromal factors with clinical significance in the ovarian tumor microenvironment

Abstract Ovarian cancer is the most lethal gynecologic malignancy in the United States. While the tumor microenvironment has been shown to play important roles in cancer pathogenesis, large-scale transcriptome profiles of the stromal component of ovarian tumors and the identification of stromal prognostic markers and therapeutic targets are lacking. This study seeks to identify secreted stromal factors and evaluate their clinical significance. Transcriptome profiling and clustering analysis on microdissected ovarian cancer associated fibroblasts (CAFs) samples from 83 patients revealed a heterogeneous gene expression pattern. Since CAFs can be derived from mesenchymal stem cells (MSCs), we compared transcriptome profiles of MSC samples obtained from healthy individuals with those from CAFs. Results showed that CAF expression profiles could be classified into two major subtypes. The MSC subtype, which has gene expression pattern highly resembling to that of undifferentiated MSCs, is significantly associated with poorer patient survival when compared to the non-MSC subtype. Further analyses identified two expression clusters within the MSC subtype: 1) the CAF-C cluster which expressed high level of MSC chondrogenic differentiation associated genes including CSPG2, SFRP2 and COMP, and 2) the CAF-O cluster which expressed high level of osteogenic differentiation associated genes including RUNX2 and BGN. Survival analysis showed that patients with the CAF-O expression subtype had overall survival rates comparable to those with the non-MSC subtypes, while the CAF-C subtype is associated with worst clinical outcomes. Pathway analyses revealed strong interactions among these stromal genes. Among them, SFRP2, which has been shown to inhibit osteogenic differentiation of MSCs by inhibiting Wnt signaling and subsequently suppress RUNX2 expression, was selected for further validation studies. Stromal SFRP2 and RUNX2 protein expression levels were evaluated by immunohistochemistry on paraffin tissue sections from 94 patients. Results showed that high levels of stromal SFRP2 were significantly associated with poor patient survival (p < 0.001), while high levels of stromal RUNX2 were significantly associated with improved patient survival (p < 0.001). A significant inverse correlation between SFRP2 and RUNX2 expression levels was also observed (r = -0.279, p = 0.037), suggested that the interactions among these genes may generate different landscapes in the tumor microenvironment, which modulate MSC differentiation and subsequently cancer cell aggressiveness. This study presents a new paradigm of which osteogenic-like and chondrogenic-like CAF signatures are associated with better and poorer patient survival respectively. Understanding the relationship between the expression patterns of stromal factors and MSC differentiation would provide us with new insights into ovarian cancer pathogenesis. Citation Format: Tsz-Lun Yeung, Cecilia S. Leung, Kwong-Kwok Wong, Samuel C. Mok. Identification and characterization of stromal factors with clinical significance in the ovarian tumor microenvironment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4799. doi:10.1158/1538-7445.AM2014-4799

Abstract 2021: A novel model to study the stroma-induced drug resistance in glioblastoma cells

Abstract Glioblastomas (GBMs) are lethal cancers with a limited response to chemotherapies. In order to study whether stromal cells convey drug resistance to glioma cells, we have established a 3 D glioma cell-specific bioluminescence assay which mimics the growth of glioma in vivo and allows for direct measurements of tumor cell viability. With lentiviral transfection experiments we have provided a panel of human glioma cell lines stably expressing the luciferase reporter gene. Luciferase expression was proportional to the number of glioma cells, but not influenced by the presence of stromal cells. This way, Luciferase signal detection could be used to monitor glioma cell viability following treatment. Glioma cells alone or cultured with stromal cells can form 3 D spheroids, then treated with Temozolomide and Doxorubicin. Through comparative analysis, stromal cells influenced the sensitivity of different chemotherapy drugs in several glioma cell lines. Experiment also proved bioluminescence assay was more sensitive than MTS assay in co-culture system. This glioma cell-specific bioluminescence platform possibly provided a tool to screen stroma-induced changes in anti-tumor drug activity. Using the platform, we will be able to screen FDA-approved drugs on different glioma cell lines and biopsies, which will potentially guide the selection of tumor associated stromal specific targets for therapeutic validation. Citation Format: Ning Yang, Huaiyang Zhu, Tao Yan, Xiao Liang, Lina Wik Leiss, Per Øyvind Enger, Xingang Li, Jian Wang. A novel model to study the stroma-induced drug resistance in glioblastoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2021. doi:10.1158/1538-7445.AM2014-2021

Abstract 4849: Implications of cancer induced blood coagulation in cancer diagnosis and therapy

Abstract A large body of clinical evidence indicates that abnormal coagulation occurs in a variety of cancers, especially invasive cancers. If cancer clusters erode adjacent normal or tumor vessels, hemorrhage may occur, and fibrin clots immediately form in situ to stop the bleeding. The fibrin clots are subsequently replaced by collagen, similar to the case in the normal wound healing process. Although there are many points in common between cancer-induced stroma and normal wound healing, the critical difference between the two is that the pathophysiological condition in cancer lasts for as long as cancer cells survive in the body (Adv Drug Deliv Rev 2012). First, we successfully developed a mAb that reacts only with human insoluble fibrin, not human fibrinogen (Cancer Sci 2011). In contrast to previously reported anti-fibrin monoclonal antibodies (mAbs), our anti-fibrin clot mAb (clone 102-10) recognizes an unexplored hole that is uncovered only when a fibrin clot forms. The epitope of the 102-10 mAb was mapped to a hydrophobic region on the Bβ-chain that interacted closely with a counterpart region on the γ-chain in the soluble state. New anti-Bβ and anti-γ mAbs specific for peptides lining the discovered hole appeared to bind exclusively to fibrin clots. Analysis of the kinetics of fibrin clot deposition using 102-10 in several disease models indicated that fibrin clot formation occurs only in the acute phase of the disease process, thrombosis, inflammation or trauma, and that these clots virtually disappear within a few weeks to be substituted by collagen. Then, we developed a new PET probe using 102-10. The radiolabelled mAb was injected into mice bearing chemically induced spontaneous tumors, and the probe showed clear and specific accumulation in the tumors. We named this detection strategy cancer stromal targeting (CAST) diagnois. Immunohistochemistry using this mAb revealed higher fibrin deposition in WHO grade 4 gliomas than in lower-grade gliomas. Because erosive tumors are highly likely to show micro-hemorrhages, even early asymptomatic tumors detected with radiolabeled 102-10 mAb may be aggressively malignant (Sci. Rep. 2013). In conclusion, CAST diagnosis and therapy based on anti-insoluble fibrin mAb may be a useful new strategy in the field of oncology. Citation Format: Yohei Hisada, Masahiro Yasunaga, Shingo Hanaoka, Shinji Saijou, Takashi Sugino, Atsushi Tsuji, Tsuneo Saga, Kouhei Tsumoto, Shino Manabe, Jun-ichiro Kuroda, Jun-ichi Kuratsu, Yasuhiro Matsumura. Implications of cancer induced blood coagulation in cancer diagnosis and therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4849. doi:10.1158/1538-7445.AM2014-4849