Significant Association of PD‐L1 With CD44 Expression and Patient Survival: Avenues for Immunotherapy and Cancer Stem Cells Downregulation in Pancreatic Cancers

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies with a propensity for metastasis and drug resistance. To overcome this and improve the outcome of patients with PDAC, there is an urgent need to elucidate the molecular mechanisms of metastasis in which cancer stem cells (CSCs) may play a role. Prrx1, a homeobox transcription factor has been identified by us, as a key molecule involved in pancreatic development, regeneration and early carcinogenesis (Reichert et al. Genes and Development 2013). Furthermore, we have reported recently that Prrx1 isoform switching governs EMT-MET plasticity to regulate PDAC invasion and metastasis (Takano et al. Genes and Development 2016). Herein, we examined that the correlation between the two Prrx1 isoforms and putative CSC markers to investigate the role of Prrx1 on pancreatic CSC functions. Materials and Methods: Mouse pancreatic ductal cell lines were isolated and established from Pdx1Cre mice (normal pancreatic ductal cells), Pdx1Cre;KrasG12D/+ mice (PanIN cells), Pdx1Cre;KrasG12D/+;p53R175H/+ mice (primary and metastatic PDAC cells). Either Prrx1a and Prrx1b was overexpressed by lentiviral transduction in each cell line with controls. Prrx1 knockdown was done in primary PDAC cells and their matched liver metastatic cells by two isoform specific siRNAs. We immunophenotyped putative pancreatic CSCs using antibodies against CSC markers (CD133, CD44, CD24 and Epcam) by flow cytometry. These subpopulations of cells were evaluated in 3D cultures, invasion assays and single-pancreatosphere formation assays. Additionally, we evaluated the expression of pancreatic CSC markers in Prrx1a and Prrx1b overexpressing and knockdown cells by qRT-PCR and flow cytometry. Results: CD133High cells and CD44HighCD24High cells are expressed highly in metastatic PDAC cells compared to primary PDAC cells. CD133High cells form spheroid cysts and CD133Low cells form spindle-shaped cells in 3D culture. CD133High cells demonstrate more invasiveness and higher self-renewal capacity compared to CD133 Low cells. The percentage of CD133High cells, CD44HighCD24High cells and EpCAMHigh cells are increased significantly in Prrx1a overexpressing primary PDAC cells. However, these cells are decreased in Prrx1a knockdown metastatic cells. Contrary to this, these subpopulation of cells are significantly decreased in Prrx1b overexpressing primary PDAC cells, and increased in Prrx1b knockdown metastatic cells. These data suggest that Prrx1a might regulate pancreatic CSC functions in metastatic PDAC cells. Conclusion: Prrx1a may be associated with a subpopulation of pancreatic cancer cells that have features of cancer stem cells. This may influence metastatic properties of pancreatic cancer cells to the liver. Citation Format: Shigetsugu Takano, Maximilian Reichert, Hideyuki Yoshitomi, Basil Bakir, Koushik K. Das, Steffen Heeg, Shingo Kagawa, Hiroaki Shimizu, Masayuki Ohtsuka, Katsunori Furukawa, Masaru Miyazaki, Anil K. Rustgi.{Authors}. Prrx1 isoforms regulate pancreatic cancer stem cell functions during pancreatic cancer progression. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B30.

Introduction: Pancreatic ductal adenocarcinoma (PDA) has the worst prognosis of all cancers and is the fourth leading cause of cancer-related deaths in the United States. Cancer stem cells (CSCs), which are a small population of tumor cells that have the ability to self-renew and differentiate into the diverse cells that comprise the tumor, have been identified in pancreatic cancer patients as lineage-negative, CD133 (prominin-1)-expressing cells. In pancreatic cancer, MUC1 is a protein expressed with a high frequency and its expression correlates with high metastasis and poor prognosis. Recent reports from our lab have demonstrated the ability of MUC1 to drive epithelial-to-mesenchymal transition thus producing a cell with ‘stem-like’ properties. We therefore investigated the correlation between MUC1 expression and CSCs. Methods: Primary tumor tissues surgically resected from patients diagnosed with pancreatic adenocarcinoma (n=8) and human pancreatic cancer cell lines were assessed for CD133 and MUC1 expression by flow cytometry. Results: In pancreatic cancer patients, tumor MUC1 levels correlated with a higher percentage of CD133-expressing cells (R=0.76). Similarly, in MiaPaCa2, Capan-1, BxPC3 WT, BxPC3 MUC1, Su86.86 WT and Su86.86 MUC1 cells lines, MUC1 levels correlated with increased CD133 expression (R=0.83). The majority of CD133+ cells isolated from primary human pancreatic tumors (82.6 ± 12.7%) expressed MUC1. Similar results were obtained using cell lines, as 83.22 ± 30.9% of CD133-expressing cells were MUC1-positive. Conclusion: MUC1 is present on the majority of CSCs and its expression correlates with higher levels of cancer stem cells. This study demonstrates yet another oncogenic role of MUC1 in pancreatic cancer progression and suggests MUC1 as a novel marker for CSCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2454. doi:10.1158/1538-7445.AM2011-2454

Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers with a 5-year survival rate less than 10%. Current therapies consisting of cytotoxic chemotherapeutic agents are not effective in most patients with metastatic PDAC, and the impact of new strategies, including immunotherapies, has not been established. Previous studies have shown that inhibition of focal adhesion kinase (FAK) modulates the PDAC immunosuppressive tumor microenvironment (TME), and sensitizes tumors to immune checkpoint blockade. However, the role of FAK in regulating tumor cell intrinsic resistance to immunotherapy is not well understood. We and others previously demonstrated FAK regulates PDAC cancer stem cells (CSCs) activity including self-renewal, tumor initiation, and drug resistance. These CSCs are responsible for metastatic dissemination and resistance to multiple therapies, including immunotherapy. In this study, we evaluated the sensitivity of PDAC cells, including CSCs to cytotoxic T cells, and we also analyzed the impact of FAK inhibition on the immunogenicity and the response to cytotoxic T cell activity of PDAC CSCs. Methods: To understand how FAK regulates tumor intrinsic resistance to antigen-specific cytotoxic T cells, we utilized OT-I T cells (OT-I cells) that specifically recognize a peptide fragment of ovalbumin (ova) within the context of MHC-I. Mouse KPC pancreatic cancer cell lines expressing ova were generated and co-cultured with cytotoxic CD8+ OT-I cells in vitro. We analyzed the effect of FAK in regulating bulk cells sensitivity to antigen-specific cytotoxic T cells and clonogenic growth of CSCs by flow cytometry, MTT assay and colony formation assay using CRISPR-Cas9 and small molecule FAK inhibitors. We also analyzed the expression of surface proteins that mediate interactions with T cells (class I MHC) in bulk cells and CSCs after FAK inhibition. Results: Loss of FAK activity decreased CSCs activity (self-renewal, colony formation) in PDAC mouse cell lines, which is consistent with our previous data. Using antigen-specific OT-I co-culture model, we found that CSCs are resistant to cytotoxic T cells anti-tumor activity compared to bulk tumor cells. We also found inhibition of FAK activity not only enhances PDAC cells to cytotoxic T cell activity, but also sensitized CSCs to T cell killing. Studies in both human and mouse PDAC cell lines demonstrated decreased MHC-I expression in CSCs compared to the bulk tumor cells which could be reversed by FAK inhibition. Conclusion: We have demonstrated that CSCs are resistant to antigen-specific cytotoxic T cells, and FAK inhibition in CSCs can sensitize these cells to anti-tumor T cell activity that is associated with changes in the expression of factors involved in antigen presentation. Our study provides novel insights into the unique immunogenicity of CSCs and the role of CSCs in regulating tumor intrinsic immunotherapy resistance in PDAC. Our findings also lead to the identification of FAK as a novel therapeutic target for CSCs to improve immunotherapy efficacy in PDAC. Citation Format: Yezi Zhu, Lyndsey Sandow, William Matsui. Inhibition of focal adhesion kinase (FAK) improves pancreatic ductal adenocarcinoma’s response to immunotherapy by targeting cancer stem cells (CSCs) [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PR-004.

Background: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with a high incidence of distant metastasis. Recent studies have shown that cancer stem cells (CSCs) are important in cancer cell growth, invasion, metastasis, and recurrence. Several studies have revealed some CSC-specific markers for PDAC, such as CD133, CD24, CD44, CXCR4, ESA, and nestin. Some evidence also suggests that PDAC progresses through a multistep process comprised of noninvasive precursor lesions known as pancreatic intraepithelial neoplasias (PanINs). The reports of CSCs in PanINs are limited. We performed a comprehensive analysis of the expression of CSC markers in PDACs, PanINs, and normal pancreatic tissues. Materials & Methods: CD24, CD44, ESA, CD133, CXCR4, and nestin were used as CSC markers. Human PDACs (n=67), PanIN-1 (n=35), PanIN-2 (n=52), PanIN-3 (n=18), and normal pancreatic tissues (n=54), which were obtained from patients who underwent surgical operations in the surgical department of Nippon Medical School, were used for immunohistochemical analysis. Human PDAC cell lines, PANC-1, KLM-1, and MIA PaCa-2, were used for flow cytometric assays and quantitative RT-PCR analysis. Results: Regarding the clinicopathological features, the positivity of CD44 and CD133 showed significant correlations for UICC classifications and histological features In the survival analysis, CD133-positive PDACs led to a significantly poorer prognosis. In the immunohistochemical analysis, CD24, CD44, ESA, CXCR4, and nestin showed a gradual increase of positivity along with the grades of PanINs. Flow cytometric assays and RT-PCR analysis confirmed the expression profile in PDAC. Conclusion: CSC markers were expressed to various extents in PDAC, and the expression of CD133 and CD44 was correlated with the prognosis and stage. Furthermore, CSC markers showed a gradual increase along with the grades of PanINs; therefore, CSC may be involved in the carcinogenesis of PDAC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 412. doi:1538-7445.AM2012-412

<div>Abstract<p>Pancreatic ductal adenocarcinoma is one of the deadliest carcinomas and is characterized by highly tumorigenic and metastatic cancer stem cells (CSC). CSCs evade available therapies, which preferentially target highly proliferative and more differentiated progenies, leaving behind CSCs as a putative source for disease relapse. Thus, to identify potentially more effective treatment regimens, we screened established and new compounds for their ability to eliminate CSCs in primary pancreatic cancer (stem) cells <i>in vitro</i> and corresponding patient-derived pancreatic cancer tissue xenografts <i>in vivo</i>. Intriguingly, we found that <i>in vitro</i> treatment with the antimalarial agent chloroquine significantly decreased CSCs, translating into diminished <i>in vivo</i> tumorigenicity and invasiveness in a large panel of pancreatic cancers. <i>In vivo</i> treatment in combination with gemcitabine was capable of more effectively eliminating established tumors and improved overall survival. The inhibitory effect of chloroquine was not related to inhibition of autophagy, but was due to inhibition of CXCL12/CXCR4 signaling, resulting in reduced phosphorylation of ERK and STAT3. Furthermore, chloroquine showed potent inhibition of hedgehog signaling by decreasing the production of Smoothened, translating into a significant reduction in sonic hedgehog-induced chemotaxis and downregulation of downstream targets in CSCs and the surrounding stroma. Our study demonstrates that via to date unreported effects, chloroquine is an effective adjuvant therapy to chemotherapy, offering more efficient tumor elimination and improved cure rates. Chloroquine should be further explored in the clinical setting as its success may help to more rapidly improve the poor prognosis of patients with pancreatic cancer. <i>Mol Cancer Ther; 13(7); 1758–71. ©2014 AACR</i>.</p></div>

Pancreatic ductal adenocarcinoma is one of the deadliest carcinomas and is characterized by highly tumorigenic and metastatic cancer stem cells (CSC). CSCs evade available therapies, which preferentially target highly proliferative and more differentiated progenies, leaving behind CSCs as a putative source for disease relapse. Thus, to identify potentially more effective treatment regimens, we screened established and new compounds for their ability to eliminate CSCs in primary pancreatic cancer (stem) cells in vitro and corresponding patient-derived pancreatic cancer tissue xenografts in vivo. Intriguingly, we found that in vitro treatment with the antimalarial agent chloroquine significantly decreased CSCs, translating into diminished in vivo tumorigenicity and invasiveness in a large panel of pancreatic cancers. In vivo treatment in combination with gemcitabine was capable of more effectively eliminating established tumors and improved overall survival. The inhibitory effect of chloroquine was not related to inhibition of autophagy, but was due to inhibition of CXCL12/CXCR4 signaling, resulting in reduced phosphorylation of ERK and STAT3. Furthermore, chloroquine showed potent inhibition of hedgehog signaling by decreasing the production of Smoothened, translating into a significant reduction in sonic hedgehog-induced chemotaxis and downregulation of downstream targets in CSCs and the surrounding stroma. Our study demonstrates that via to date unreported effects, chloroquine is an effective adjuvant therapy to chemotherapy, offering more efficient tumor elimination and improved cure rates. Chloroquine should be further explored in the clinical setting as its success may help to more rapidly improve the poor prognosis of patients with pancreatic cancer.

<div>Abstract<p>Pancreatic ductal adenocarcinoma is one of the deadliest carcinomas and is characterized by highly tumorigenic and metastatic cancer stem cells (CSC). CSCs evade available therapies, which preferentially target highly proliferative and more differentiated progenies, leaving behind CSCs as a putative source for disease relapse. Thus, to identify potentially more effective treatment regimens, we screened established and new compounds for their ability to eliminate CSCs in primary pancreatic cancer (stem) cells <i>in vitro</i> and corresponding patient-derived pancreatic cancer tissue xenografts <i>in vivo</i>. Intriguingly, we found that <i>in vitro</i> treatment with the antimalarial agent chloroquine significantly decreased CSCs, translating into diminished <i>in vivo</i> tumorigenicity and invasiveness in a large panel of pancreatic cancers. <i>In vivo</i> treatment in combination with gemcitabine was capable of more effectively eliminating established tumors and improved overall survival. The inhibitory effect of chloroquine was not related to inhibition of autophagy, but was due to inhibition of CXCL12/CXCR4 signaling, resulting in reduced phosphorylation of ERK and STAT3. Furthermore, chloroquine showed potent inhibition of hedgehog signaling by decreasing the production of Smoothened, translating into a significant reduction in sonic hedgehog-induced chemotaxis and downregulation of downstream targets in CSCs and the surrounding stroma. Our study demonstrates that via to date unreported effects, chloroquine is an effective adjuvant therapy to chemotherapy, offering more efficient tumor elimination and improved cure rates. Chloroquine should be further explored in the clinical setting as its success may help to more rapidly improve the poor prognosis of patients with pancreatic cancer. <i>Mol Cancer Ther; 13(7); 1758–71. ©2014 AACR</i>.</p></div>

Finding and Killing the CRABs of Pancreatic Cancer

Background: Cancer stem cells (CSCs) have been described to confer resistance and aggressive disease biology in pancreatic ductal adenocarcinoma (PDAC) and ampullary carcinoma (AMPC). Patient derived cancer organoids (PCOs) are an important model of PDAC that maintain the molecular features of advanced disease and have been shown to predict drug response. This study aimed to determine the dynamics of CSC expression on the oligoclonal culture growth of three independent PCOs models. Methods: PCOs were assayed using low volume 10μL microformat in 96 well design (Ibidi, Inc). PCOs were treated with physiologic FOLFIRINOX or gemcitabine + nab-paclitaxel (nab-pac) including single agent controls. Response was assayed by growth using timecourse data from 0h to 72h using high content imaging (Cytation 5). At 72h, the PCOs were stained using four distinct CSC markers (CD44, CD133, CD117, and EpCAM). Images were aligned using thresholded root mean standard deviation of 75μm and tracked for normalized change in longest diameter. At 72h, CSC markers were tracked using % area positive from brightfield region of interest. Response was tracked by effect size Glass’s Delta (GΔ) defined as the difference between the mean of media control and treatment group normalized to standard deviation of media control. Results: PCOs maintained nuclear atypia and high nuclear-chromatin ratios consistent with matured xenografts. AMPC1 showed similar sensitivity with growth arrest by PCO Δ diameter for gem + nab-pac (mean = +2.2%, GΔ = 1.29) and FOLFIRINOX (mean = 0.0%, GΔ = 1.33). AMPC1 had CSC enrichment for CD44 after treatment with nab-pac (GΔ = 0.93), CD133 expression after treatment with gem + nab-pac (GΔ = 0.75), CD117 after treatment with nab-pac treatment (GΔ = 0.80), and EpCAM after treatment with gem + nab-pac (GΔ = 0.78). In AMPC1, interquartile analysis revealed increased CSC expression conferred reduced growth for CD133 (+72% v. 37%, p<0.005) and CD117 (+81% v. +25%, p<0.005). Normalized Δ diameter across PDAC PCOs revealed PDAC1 had reduced sensitivity to gem + nab-pac (mean = +16.0%, GΔ = 1.2) vs. PDAC2 (Mean = 2.1%, GΔ = 1.65). PDAC1 had CSC enrichment with increased % area of CD44+ after treatment with gem + nab-pac (GΔ = 1.13). In PDAC2, interquartile analysis revealed increased CSC expression conferred decreased growth rate for CD133 (+65% v. +36%, p< 0.005) and CD117 (+75% v. +38%, p<0.005). Conclusions: PDAC and AMPC PCOs reveal oligoclonal expression of CSC markers within a population. Increased CSC expression for CD133 and CD117 within a population consistently revealed decreased growth within an organoid population. CSC expression (CD44, CD133, CD117, and EpCAM) was found to be enriched under therapeutic challenge with gem and nab-pac containing chemotherapy. This work supports the oligoclonal and dynamic nature of CSCs for pancreaticobiliary cancers in response to therapeutic challenge. Citation Format: Eleanor Riedl, Austin Stram, MD Shahadat Hossan, Ethan S. Lin, Luke Koeppel, Jamie Warner, Sam Lubner, Stephanie McGregor, Wei Zhang, Jeremy Kratz. Tracking cancer stem cell expression of pancreatic and ampullary cancer organoids in response to chemotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5809.

Introduction: Recent studies have demonstrated a clear association between smoking and the incidence of pancreatic ductal adenocarcinoma (PDAC); however, the effect of cigarette smoke in the activation of stem cell (SC) or cancer stem cell (CSC) genes and their involvement in the initiation and progression of PDAC have not yet been studied. It is well known that CSCs are responsible for the drug resistance and aggressiveness of the disease including PDAC. In this study, we investigated the effects of smoking on enrichment of SC/CSCs in pancreatic normal and ductal adenocarcinoma cells, and we also examined whether smoking can activate NF-kB signaling, which is in part leads to enrichment of CSC and induction of CSC markers in PDAC. Methods: Cigarette smoke extract (CSE) was prepared, and HPNE (Human pancreatic nestin positive cells) and Capan-1 pancreatic cancer (PC) cells were treated with CSE for up to ∼15 weeks. Side population (SP) were analyzed by Hoechst staining using Flow-cytomer, and various CSC markers such as PD2 (a stem cell maintenance marker), CD44, ALDH-1, SOX-9 (a multipotent SC marker) and Oct-3/4 (a pluripotent marker), and NF-kB signaling molecules were analyzed by western blotting. ALDH1+ cells, CD44+CD24+ CSCs and G0/G1 phase low cycling quiescent cells were analyzed by flow cytometer. An in-vitro sphere culture was also performed to further confirm the smoke induced CSC properties. Smoke exposed pancreatic tissues excised from unfloxed littermate control (LSL-K-Ras G12D) pancreatic tissue sections were immunostained for SOX-9 using immunohistochemistry (IHC), and for SOX9 and CD44 using immunofluorescence. Results: Our results showed increased SC/CSCs and more number of spheres by CSE treated cells as compared to their untreated controls and displayed elevated protein expressions of SC/CSC markers. We also observed an elevated CD44+CD24+ CSCs, increased ALDH1+ cells and increased G0/G1 low cycling quiescent cell population in CSE treated cells as compared to untreated controls. In addition, increased immunohistochemical staining for SOX9 and increased immunofluorescent signal for SOX-9 and CD44 were observed in smoke exposed animal tissues indicating that smoking may transform SOX-9+ multipotent SCs into SOX9+CD44+ CSCs. We also analyzed the expression levels of NF-kB signaling molecules in CSE treated HPNE and Capan-1 cells. As compared to their untreated controls, CSE treated cells showed elevated protein expression levels of phospho AKT, phospho RelA (SOX-9 promoter binding subunit of NF-kB complex) and phospho IKKα (a kinase that phosphorylates IKBα, an inhibitor of RelA) suggesting that smoking activates NF-kB signaling. Conclusion: Our results illustrate that smoking enriches SC/CSCs populations in normal pancreatic cells as well as in pancreatic cancer cells, and activates SOX9 through NF-kB signaling in PDAC. Citation Format: Ramakrishna Nimmakayala, Parthasarathy Seshacharyulu, Seema Chugh, Imayavaramban Lakshmanan, Satyanarayana Rachagani, Surinder K Batra, Moorthy P Ponnusamy. Smoking enriches cancer stem cell population and activates Sox9 through NF-kB signaling in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2490.

<div>Abstract<p><b>Purpose:</b> Pancreatic ductal adenocarcinoma (PDAC) is a cancer of the exocrine pancreas with unmet medical need and is strongly promoted by tumor-associated macrophages (TAM). The presence of TAMs is associated with poor clinical outcome, and their overall role, therefore, appears to be protumorigenic. The “don't eat me” signal CD47 on cancer cells communicates to the signal regulatory protein-α on macrophages and prevents their phagocytosis. Thus, inhibition of CD47 may offer a new opportunity to turn TAMs against PDAC cells, including cancer stem cells (CSC), as the exclusively tumorigenic population.</p><p><b>Experimental Design:</b> We studied <i>in vitro</i> and <i>in vivo</i> the effects of CD47 inhibition on CSCs using a large set of primary pancreatic cancer (stem) cells as well as xenografts of primary human PDAC tissue.</p><p><b>Results:</b> CD47 was highly expressed on CSCs, but not on other nonmalignant cells in the pancreas. Targeting CD47 efficiently enhanced phagocytosis of a representative set of primary human pancreatic cancer (stem) cells and, even more intriguingly, also directly induced their apoptosis in the absence of macrophages during long-term inhibition of CD47. In patient-derived xenograft models, CD47 targeting alone did not result in relevant slowing of tumor growth, but the addition of gemcitabine or Abraxane resulted in sustained tumor regression and prevention of disease relapse long after discontinuation of treatment.</p><p><b>Conclusions:</b> These data are consistent with efficient <i>in vivo</i> targeting of CSCs, and strongly suggest that CD47 inhibition could be a novel adjuvant treatment strategy for PDAC independent of underlying and highly variable driver mutations. <i>Clin Cancer Res; 21(10); 2325–37. ©2015 AACR</i>.</p></div>

<div>Abstract<p><b>Purpose:</b> Pancreatic ductal adenocarcinoma (PDAC) is a cancer of the exocrine pancreas with unmet medical need and is strongly promoted by tumor-associated macrophages (TAM). The presence of TAMs is associated with poor clinical outcome, and their overall role, therefore, appears to be protumorigenic. The “don't eat me” signal CD47 on cancer cells communicates to the signal regulatory protein-α on macrophages and prevents their phagocytosis. Thus, inhibition of CD47 may offer a new opportunity to turn TAMs against PDAC cells, including cancer stem cells (CSC), as the exclusively tumorigenic population.</p><p><b>Experimental Design:</b> We studied <i>in vitro</i> and <i>in vivo</i> the effects of CD47 inhibition on CSCs using a large set of primary pancreatic cancer (stem) cells as well as xenografts of primary human PDAC tissue.</p><p><b>Results:</b> CD47 was highly expressed on CSCs, but not on other nonmalignant cells in the pancreas. Targeting CD47 efficiently enhanced phagocytosis of a representative set of primary human pancreatic cancer (stem) cells and, even more intriguingly, also directly induced their apoptosis in the absence of macrophages during long-term inhibition of CD47. In patient-derived xenograft models, CD47 targeting alone did not result in relevant slowing of tumor growth, but the addition of gemcitabine or Abraxane resulted in sustained tumor regression and prevention of disease relapse long after discontinuation of treatment.</p><p><b>Conclusions:</b> These data are consistent with efficient <i>in vivo</i> targeting of CSCs, and strongly suggest that CD47 inhibition could be a novel adjuvant treatment strategy for PDAC independent of underlying and highly variable driver mutations. <i>Clin Cancer Res; 21(10); 2325–37. ©2015 AACR</i>.</p></div>

Solid tumors display a complex biology that requires a multipronged treatment strategy. Most anticancer interventions, including chemotherapy, are currently unable to prevent treatment resistance and relapse. In general, therapeutics target cancer cells and overlook the tumor microenvironment (TME) and the presence of cancer stem cells (CSCs) with self-renewal and tumorigenic abilities. CSCs have been postulated to play key roles in tumor initiation, progression, therapy resistance, and metastasis. Hence, CSC markers have been suggested as diagnostics to forecast cancer prognosis as well as molecular targets for new-generation cancer treatments, especially in resistant disease. We report here original findings on expression and prognostic significance of CSC markers in several cancers. We examined and compared the transcriptional expression of CSC markers (ABCB1, ABCG2, ALDH1A1, CD24, CD44, CD90, CD133, CXCR4, EPCAM, ICAM1, and NES) in tumor tissues versus the adjacent normal tissues using publicly available databases, The Cancer Genome Atlas and Gene Expression Profiling Interactive Analysis. We found that CSC transcriptional markers were, to a large extent, expressed in higher abundance in solid tumors such as colon, lung, pancreatic, and esophageal cancers. On the other hand, no CSC marker in our analysis was expressed in the same pattern in all cancers, while individual CSC marker expression, alone, was not significantly associated with overall patient survival. Innovation in next-generation cancer therapeutics and diagnostics ought to combine CSC markers as well as integrative diagnostics that pool knowledge from CSCs and other TME components and cancer cells.

Background: Cancer stem cells (CSCs) refer to a subset of tumor cells that have self-renewal ability and generate plenty of non-CSC cells that comprise a tumor. In addition, CSCs play crucial roles in various processes during tumor progression and metastasis as well. A number of CSC markers candidates have been explored to date, and Aldehyde dehydrogenase 1 (ALDH1), c-Met, and CD44 have been identified as CSC markers in pancreatic ductal adenocarcinoma (PDAC). On the other hand, prostaglandin E2 (PGE2) is one of metabolites in arachidonate cascade and is implicated in the expansion of hematopoietic and tissue stem cell fraction. The aim of this study is to single out the most important CSC marker and elucidate the functional role of PGE2 for CSC expansion in PDAC. Methods: Three CSC markers (ALDH1, CD44, and c-Met) expression was examined by immunohistochemistry in 121 primary surgical specimens of PDAC and analyzed a relationship with clinicopathological factors and clinical outcomes. The clonogenic growth potential of CSC marker-positive PDAC cells was assessed in vitro by growth assays and sphere formation assays. We next investigated the expression of CSC markers and self-renewal related genes in PDAC cell lines with PGE2 or 15-PGDH inhibitor treatment. We further conducted functional experiments using siRNA to identify the critical molecule in PDAC progression. Results: A high level of ALDH1 expression was detected in 63 of the 121 cases, and was significantly associated with large tumor size and poor prognosis in PDAC patiants. On the other hand, CD44 and c-Met expression were not associated with the prognosis. Among CSC markers, the expression of ALDH1 was significantly increased by PGE2 treatment in PDAC cells. By suppressing ALDH1 expression by siRNA, growth and sphere formation potential were inhibited in ALDH1 high-expressing PDAC cells. In contrast, the expression of ALDH1 was remarkably increased by PGE2 or 15-PGDH inhibitor treatment in ALDH1 low-expressing PDAC cells. Finally, we found that Nanog and Oct-4 were down-stream molecules of PGE2-ALDH1 signaling and played crucial roles for PDAC cell expansion. Conclusion: Our results demonstrated that PGE2 positively regulated ALDH1 expression, and the growth and sphere formation potential were promoted by increasing ALDH1 expression, resulting in poor prognosis of PDAC patients. Inhibiting PGE2-ALDH1 signaling could lead to the suppression of tumor growth in PDAC patients. Citation Format: Kota Arima, Takatsugu Ishimoto, Hirohisa Okabe, Yuki Kitano, Risa Inoue, Kensuke Yamamura, Takayoshi Kaida, Takaaki Higashi, Katsunobu Taki, Katsunori Imai, Daisuke Hashimoto, Akira Chikamoto, Toru Beppu, Hideo Baba. Verification of mechanism that CSC markers are implicated in poor prognosis for pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3350.

Pancreatic Ductal Adenocarcinoma (PDAC) is the most common type of pancreatic malignancies. It is known for its aggressive nature and high mortality rate. This calls for an urgent need of new prognostic and therapeutic markers that can be targeted for personalized treatment of the patient. Among 142 patients diagnosed with pancreatic cancers at Aga Khan University Hospital, a total of 62 patients were selected based on their confirmed diagnosis of PDAC. Immunohistochemistry was performed on Formalin-Fixed Paraffin-Embedded (FFPE) sections using selected antibodies (CD44, CD133, L1CAM, HER2, PD-L1, EGFR, COX2 and cyclin D1). All the slides were scored independently by two pathologists as per the set criteria. Expression of all cancer stem cell markers was found to be significantly associated with one or more potential therapeutic markers. CD44 expression was significantly associated with HER2 (p = 0.032), COX2 (p = 0.005) and EGFR expression (p = 0.008). CD133 expression also showed significant association with HER2 (p = 0.036), COX2 (p = 0.004) and EGFR expression (p = 0.018). L1CAM expression was found to be associated with expression of COX2 (p = 0.017). None of the proteins markers showed association with overall survival of the patient. On the other hand, among the clinicopathological characteristics, histological differentiation (p = 0.047), lymphovascular invasion (p = 0.021) and perineural invasion (p = 0.014) were found to be significantly associated with patient's overall survival. Internationally, this is the first report that assesses the selected panel of cancer stem cell markers and potential therapeutic targets in a single study and evaluates its combined expression. The study clearly demonstrates association between expression of cancer stem cell markers and therapeutic targets hence paves a way for precision medicine for pancreatic cancer patients.