Human Pancreatic Cancer Xenografts Research Articles

DBDx-based drug combinations show highly potent therapeutic efficacy against human pancreatic cancer xenografts in athymic mice

ABSTRACT Previous studies have shown that DBDx, a combination consisting of dipyridamole, bestatin and dexamethasone is highly effective against several cancer xenografts in athymic mice. Here the therapeutic effects of DBDx and its combination with gemcitabine or capcitabine against human pancreatic cancer xenografts and the mechanism were studied. In vivo experiments performed in athymic mice showed that the antitumor efficacy of DBDx was much stronger than that of gemcitabine or capecitabine alone. Notably, the combination of DBDx and gemcitabine or capcitabine further enhanced the efficacy. In the case of DBDx (242 mg/kg) plus gemcitabine (100 mg/kg), tumor weight decreased about 97.7%, and tumor sizes were shrinking during the treatment. In the case of DBDx (242 mg/kg) plus capecitabine (718.7 mg/kg), tumor weight decreased about 94.9%. Moreover, DBDx and its combinations obviously prolonged theoverall survival of mice compared with gemcitabine or capcitabine alone. DBDx-based drug combination therapy showed no obvious systematic toxicity. The gene expression profile analysis showed that the genes changed by DBDx were related to immune system and tumor vasculature. The result of protein array showed that the changed proteins in the serum of treated mice were related to immune and inflammation system. These results show that DBDx-based drug combinations, a new strategy which integrates the use of low-cytotoxic drugs and cytotoxic chemotherapeutics, are highly effective regimens against human pancreatic cancer in athymic mice at well tolerated doses. DBDx-based drug combination therapy might provide new options for the treatment of pancreatic cancer.

Radioimmunotherapy of human pancreatic cancer xenografts in NOD-scid mice with [64Cu]Cu-NOTA-panitumumab F(ab′)2 alone or combined with radiosensitizing gemcitabine and the PARP inhibitor, rucaparib

IntroductionOur objective was to determine the feasibility of extending our previously reported PET imaging study of pancreatic cancer (PnCa) with [64Cu]Cu-NOTA-panitumumab F(ab′)2 to radioimmunotherapy (RIT) by exploiting the β-particle and Auger electron emissions of 64Cu (PET theranostic concept). To enhance the effectiveness of [64Cu]Cu-NOTA-panitumumab F(ab′)2, we further combined RIT with radiosensitizing gemcitabine (GEM) and the poly(ADP)ribose polymerase inhibitor (PARPi), rucaparib. MethodsNormal tissue toxicity was assessed in non-tumor-bearing NOD-scid mice injected i.v. with [64Cu]Cu-NOTA-panitumumab F(ab′)2 (1.85–9.25 MBq; 10 μg) or [64Cu]Cu-NOTA-anti-mouse EGFR Ab30 F(ab′)2 (12.95 MBq). Body weight was monitored, and hematopoietic (CBC), liver (ALT) and kidney [creatinine (SCr)] toxicity were assessed. RIT studies were performed in NOD-scid mice with s.c. OCIP23 human PnCa patient-derived xenografts (PDX) administered [64Cu]Cu-NOTA-panitumumab F(ab′)2 (3.7 MBq; 10 μg), unlabeled panitumumab F(ab′)2 (10 μg) or normal saline every two weeks. Subsequent studies evaluated RIT with [64Cu]Cu-NOTA-panitumumab F(ab′)2 (12.95 MBq; 10 μg) administered alone or combined with GEM and the PARPi, rucaparib administered on a 14-day treatment cycle for up to 6 cycles in NOD-scid mice with s.c. PANC-1 human PnCa xenografts. The radiation absorbed dose in PANC-1 tumors and normal organs in mice after a single i.v. injection of [64Cu]Cu-NOTA-panitumumab F(ab′)2 (12.95 MBq; 10 μg) was estimated based on previously reported biodistribution studies of [64Cu]Cu-NOTA-panitumumab F(ab′)2. ResultsNo normal tissue toxicity was observed in non-tumor-bearing NOD-scid mice administered up to 3.7 MBq (10 μg) of [64Cu]Cu-NOTA-panitumumab F(ab′)2 but slightly increased ALT was noted at 9.25 MBq. Administration of [64Cu]Cu-NOTA-anti-mouse EGFR Ab30 F(ab′)2 (12.95 MBq; 10 μg) caused some hematopoietic toxicity but no increase in ALT or SCr or decreased body weight. A slight tumor growth delay and increased survival was noted in NOD-scid mice with s.c. OCIP23 PDX treated with [64Cu]Cu-NOTA-panitumumab F(ab′)2 (3.7 MBq; 10 μg) or unlabeled panitumumab F(ab′)2 (10 μg) compared to normal saline treated mice. RIT with [64Cu]Cu-NOTA-panitumumab F(ab′)2 (12.95 MBq; 10 μg) combined with GEM + PARPi for up to 6 cycles was most effective for the treatment of PANC-1 tumors. Tumor doubling time increased to 13.3 ± 0.9 days vs. 7.8 ± 3.7 days for RIT alone and 9.3 ± 2.2 days for normal saline treatment. Median survival was significantly longer (P < 0.05) than in mice treated with normal saline (35 days) for RIT + GEM + PARPi (71 days), GEM + PARPi (44 days) and RIT + GEM (43 days) but not for RIT alone (25 days). RIT + GEM + PARPi provided a longer median survival than RIT (P < 0.01), GEM + PARPi (P = 0.01) but not RIT + GEM (P = 0.23). Nonetheless, PANC-1 tumors grew exponentially in all treatment groups. The absorbed dose in PANC-1 tumors after a single i.v. injection of [64Cu]Cu-NOTA-panitumumab F(ab′)2 (12.85 MBq; 10 μg) was 0.8 Gy, while the dose in normal organs ranged from 0.6–1.2 Gy. ConclusionsWe conclude that RIT with [64Cu]Cu-NOTA-panitumumab F(ab′)2 did not cause significant normal tissue toxicity but was not effective when administered alone for treatment of PnCa xenografts in NOD-scid mice. Combining RIT with GEM and the PARPi, rucaparib enhanced its effectiveness but tumors continued to grow exponentially. Our results suggest that 64Cu is not feasible for RIT of PnCa due to low tumor absorbed doses. 177Lu which has a higher abundance of moderate energy β-particle emissions may be more effective than 64Cu. The hematopoietic toxicity of [64Cu]Cu-NOTA-anti-mouse EGFR Ab30 F(ab′)2 may be mediated by binding to mouse EGFR expressed on some hematopoietic stem cells. Advances in knowledge and implications for patient careDirect extension of PET with 64Cu(Cu)-NOTA-panitumumab F(ab′)2 to RIT exploiting the β-particle and Auger electron emissions of 64Cu is not feasible. Theranostic approaches that combine PET with RIT employing 177Lu may be more promising and should be explored.

Exploiting Arginine Auxotrophy with Pegylated Arginine Deiminase (ADI-PEG20) to Sensitize Pancreatic Cancer to Radiotherapy via Metabolic Dysregulation.

Distinct metabolic vulnerabilities of cancer cells compared with normal cells can potentially be exploited for therapeutic targeting. Deficiency of argininosuccinate synthetase-1 (ASS1) in pancreatic cancers creates auxotrophy for the semiessential amino acid arginine. We explored the therapeutic potential of depleting exogenous arginine via pegylated arginine deiminase (ADI-PEG20) treatment as an adjunct to radiotherapy. We evaluated the efficacy of treatment of human pancreatic cancer cell lines and xenografts with ADI-PEG20 and radiation via clonogenic assays and tumor growth delay experiments. We also investigated potential mechanisms of action using reverse-phase protein array, Western blotting, and IHC and immunofluorescence staining. ADI-PEG20 potently radiosensitized ASS1-deficient pancreatic cancer cells (MiaPaCa-2, Panc-1, AsPc-1, HPAC, and CaPan-1), but not ASS1-expressing cell lines (Bxpc3, L3.6pl, and SW1990). Reverse phase protein array studies confirmed increased expression of proteins related to endoplasmic reticulum (ER) stress and apoptosis, which were confirmed by Western blot analysis. Inhibition of ER stress signaling with 4-phenylbutyrate abrogated the expression of ER stress proteins and reversed radiosensitization by ADI-PEG20. Independent in vivo studies in two xenograft models confirmed significant tumor growth delays, which were associated with enhanced expression of ER stress proteins and apoptosis markers and reduced expression of proliferation and angiogenesis markers. ADI-PEG20 augmented the effects of radiation by triggering the ER stress pathway, leading to apoptosis in pancreatic tumor cells.

1,2,3-Triazolyl ester of ketorolac (15K), a potent PAK1 blocker, inhibits both growth and metastasis of orthotopic human pancreatic cancer xenografts in mice.

More than 90% of human pancreatic cancers carry the oncogenic mutant of Ki-RAS and their growth depends on its downstream kinase PAK1, mainly because PAK1 blocks the apoptosis of cancer cells selectively. We developed a highly cell-permeable PAK1-blocker called 15K from an old pain-killer (ketorolac), that is shown here to inhibit the growth of three pancreatic cancer cell lines with IC50 values ranging 41-88 nM in vitro. The anti-cancer effect of 15K was further investigated in an orthotopic xenograft model with gemcitabine (GEM)-resistant human pancreatic cancer cell lines (AsPC-1 and BxPC-3) expressing luciferase in athymic mice. During 4 weeks, 15K blocks total burden (growth) of both AsPC-1 and BxPC-3 tumors (measured as radians/sec) with the IC50 below daily dose of 0.1 mg/kg, i.p. In a similar manner 15K reduced both their invasion and metastases as well, while it had no effect on either body weight or hematological parameters even at 5 mg/kg/day. To the best of our knowledge, 15K is so far the most potent among synthetic PAK1-blockers in vivo, and could be potentially useful for therapy of GEM-resistant cancers.

Theranostics Targeting Fibroblast Activation Protein in the Tumor Stroma: 64Cu- and 225Ac-Labeled FAPI-04 in Pancreatic Cancer Xenograft Mouse Models.

Fibroblast activation protein (FAP), which promotes tumor growth and progression, is overexpressed in cancer-associated fibroblasts of many human epithelial cancers. Because of its low expression in normal organs, FAP is an excellent target for theranostics. In this study, we used radionuclides with relatively long half-lives, 64Cu (half-life, 12.7 h) and 225Ac (half-life, 10 d), to label FAP inhibitors (FAPIs) in mice with human pancreatic cancer xenografts. Methods: Male nude mice (body weight, 22.5 ± 1.2 g) were subcutaneously injected with human pancreatic cancer cells (PANC-1, n = 12; MIA PaCa-2, n = 8). Tumor xenograft mice were investigated after the intravenous injection of 64Cu-FAPI-04 (7.21 ± 0.46 MBq) by dynamic and delayed PET scans (2.5 h after injection). Static scans 1 h after the injection of 68Ga-FAPI-04 (3.6 ± 1.4 MBq) were also acquired for comparisons using the same cohort of mice (n = 8). Immunohistochemical staining was performed to confirm FAP expression in tumor xenografts using an FAP-α-antibody. For radioligand therapy, 225Ac-FAPI-04 (34 kBq) was injected into PANC-1 xenograft mice (n = 6). Tumor size was monitored and compared with that of control mice (n = 6). Results: Dynamic imaging of 64Cu-FAPI-04 showed rapid clearance through the kidneys and slow washout from tumors. Delayed PET imaging of 64Cu-FAPI-04 showed mild uptake in tumors and relatively high uptake in the liver and intestine. Accumulation levels in the tumor or normal organs were significantly higher for 64Cu-FAPI-04 than for 68Ga-FAPI-04, except in the heart, and excretion in the urine was higher for 68Ga-FAPI-04 than for 64Cu-FAPI-04. Immunohistochemical staining revealed abundant FAP expression in the stroma of xenografts. 225Ac-FAPI-04 injection showed significant tumor growth suppression in the PANC-1 xenograft mice, compared with the control mice, without a significant change in body weight. Conclusion: This proof-of-concept study showed that 64Cu-FAPI-04 and 225Ac-FAPI-04 could be used in theranostics for the treatment of FAP-expressing pancreatic cancer. α-therapy targeting FAP in the cancer stroma is effective and will contribute to the development of a new treatment strategy.

A Novel Oxoglutarate Dehydrogenase-Like Mediated miR-214/TWIST1 Negative Feedback Loop Inhibits Pancreatic Cancer Growth and Metastasis.

As a main rate-limiting subunit of the 2-oxoglutarate dehydrogenase multienzyme complex, oxoglutarate dehydrogenase like (OGDHL) is involved in the tricarboxylic acid cycle, and frequently downregulated in human carcinoma and suppresses tumor growth. However, little is known about the role of OGDHL in human cancer, especially pancreatic cancer. Our goal is to study the underlying mechanism and define a novel signaling pathway controlled by OGDHL modulating pancreatic cancer progression. The expression and functional analysis of OGDHL, miR-214, and TWIST1 in human pancreatic cancer tissues, cell lines, and xenograft tumor model were investigated. The correlations between OGDHL and those markers were analyzed. OGDHL was downregulated in human pancreatic cancer and predicted poor prognosis. OGDHL overexpression inhibited migration and invasion of pancreatic cancer cells and suppressed pancreatic cancer tumor growth. OGDHL was shown to be negatively regulated by miR-214. TWIST1 upregulation induced miR-214 expression in pancreatic cancer. OGDHL suppressed TWIST1 expression through promoting ubiquitin-mediated proteasomal degradation of HIF1α and regulating AKT pathways. A combination of OGDHL downregulation and TWIST1 and miR-214 overexpression predicted worse prognosis in patients with pancreatic cancer. We demonstrated the prognostic value of OGDHL, miR-214, and TWIST1 in pancreatic cancer, and elucidated a novel pathway in OGDHL-regulated inhibition of pancreatic cancer tumorigenesis and metastasis. These findings may lead to new targeted therapy for pancreatic cancer through regulating OGDHL, miR-214, and TWIST1.

Abstract 5265: Spleen metabolism altered by human pancreatic cancer xenografts

Abstract Cancer-induced cachexia accounts for approximately 20% of all cancer deaths [1] and affects nearly 80% of pancreatic cancer patients [2, 3]. Cachectic patients experience a wide range of symptoms affecting several organ functions such as muscle, liver, brain, and heart that decrease quality of life and worsen prognosis. A major characteristic of cachexia is the accelerated skeletal muscle and fat storage wasting causing nutrient mobilization both directly as lipid and amino acids, and indirectly as glucose derived from the exploitation of liver gluconeogenesis that reaches the tumor through the bloodstream [4]. Here, for the first time, we have performed high-resolution quantitative 1H magnetic resonance spectroscopy (MRS) of spleen tissue obtained from normal mice and mice bearing PDAC that are cachectic (Pa04C) and non-cachectic (Panc1). The Panc1 (non-cachectic), and Pa04C (cachectic) PDAC cell lines were used [5]. Eight-week-old male severe combined immunodeficient mice were inoculated in the right flank with cancer cells (5×106). Mice were euthanized once tumors were ~ 300 mm3. Control, cachectic and non-cachectic groups consisted of 9, 10 and 9 mice per group respectively. Spleen water soluble metabolites were extracted, freeze dried, reconstituted in D2O PBS and transferred to an NMR tube for spectral acquisition with a 750 MHz (17.6T) MR spectrometer [6]. As anticipated, mice with cachexia-inducing Pa04C tumors showed significant weight loss with time. We observed, for the first time, that spleens from cachectic Pa04C tumor bearing mice showed a profound weight loss compared to normal mice and mice with Panc1 tumors. However, an increase of liver and spleen size has been previously reported in terminal cachectic human patients from colorectal cancer measured by CT scan 2-11 months prior to death [7]. A significant decrease in almost all amino acids was observed in cachectic (Pa04C) mouse spleens compared to normal and non-cachectic (Panc1) mouse spleens. Differences in choline metabolites, creatine, glutamine, glutamate, glutathione and aspartate were observed in cachectic mouse spleens compared to non-cachectic mouse spleens and spleens from healthy control mice. The significant decrease of amino acids in the cachectic spleens may reflect increased utilization of amino acids by the tumor or other organs during the cachexia muscle/protein wasting [4]. These results provide new insights into changes in spleen metabolism during cachexia, and support investigating metabolic targets to reduce cachexia associated morbidity. Supported by NIH R01CA193365 and R35CA209960. Reference: 1. Argiles et al. Nat. Rev. Cancer 2014, 14 (11), 754; 2. Fearon et al. HPB (Oxford) 2010, 12 (5), 323; 3. Ozola et al. Pancreatology 2015, 15 (1), 19; 4. Porporato et al. Oncogenesis 2016, 5, e200; 5. Winnard et al. Can. Res. 2016, 76(6): 1441; 6. Penet et al. Clin. Can. Res. 2015, 21 (2), 386; 7. Lieffers et al. Am J Clin Nutr. 2009, 89 (4), 1173 Citation Format: Santosh Kumar Bharti, Paul T. Winnard, Raj Kumar Sharma, Yelena Mironchik, Marie-France Penet, Zaver M. Bhujwalla. Spleen metabolism altered by human pancreatic cancer xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5265.

Abstract 3498: Highly specific macrocyclic ATR inhibitors for the targeted treatment of a broad spectrum of cancers showing lack of anemia or neutropenia in pre-clinical animal models

Abstract Ataxia Telangiectasia and Rad3-related (ATR) and its downstream effector Checkpoint Kinase 1 (CHK1) are central to the protection of stalled replication forks.Specific targeting of the ATR is synthetically lethal with multiple cancer-associated changes including oncogenic stress and defects in the DDR pathway and represents an emerging strategy to treat a broad spectrum of cancers. Atrin Pharmaceuticals has rationally designed a novel series of conformationally constrained macrocyclic ATR inhibitors with higher potency and selectivity than other ATR inhibitors currently in clinical development. An in-depth characterization of the ATRN series identified ATRN-119 as our lead compound with an in vitro enzyme IC50 of 20 nM and inhibition of ATR substrate CHK1 Ser345 phosphorylation in cells at an IC50 of 5 nM. ATRN-119 inhibits ATR in cells at much lower concentrations and demonstrates higher selectivity (&amp;gt;2000) for ATR over other closely-related PIK-kinases like ATM, DNA-PK and mTOR. Additionally, ATRN series have favorable ADME properties with increased water solubility and metabolic stability in human serum of up to 4 hours. Oral dosing of ATRN-119 showed significant antitumor effects in human pancreatic and colon cancer xenografts and in orthotopic ovarian Patient Derived Xenograft (PDX) tumors. Notably, hematological analysis of mice treated with daily oral dosing of ATRN-119 indicated no thrombocytopenia, anemia or neutropenia up to 4 weeks of treatment. Exploratory multiple high dosing toxicity studies in rats and dogs indicate significant exposure and good tolerability with lack of anemia or neutropenia. In a series of in-vitro cell viability assays, three dimensional organoid cultures and in-vivo combinationstudies, ATRN-119 showed significant synergism with various PARP inhibitors as well as restoration of PARPi sensitivity in PARPi resistant tumors. Our data suggests a new generation of highly potent and selective ATR inhibitors with favorable safety profile and a broad clinical therapeutic potential either as monotherapy, in combination with PARP inhibitors or as a synthetic lethal approach with key DDR mutations. Citation Format: Sahithi Pamarthy, Dansu Li, Ekaterine Goliadze, Tina Gill, Lanqi Jia, Erin George, Laura R. Butler, Ryan L. Ragland, Michel Afargan, Fiona A. Simpkins, Eric J. Brown, Oren Gilad. Highly specific macrocyclic ATR inhibitors for the targeted treatment of a broad spectrum of cancers showing lack of anemia or neutropenia in pre-clinical animal models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3498.

Selective activation of tumor-suppressive MAPKP signaling pathway by triptonide effectively inhibits pancreatic cancer cell tumorigenicity and tumor growth

The mitogen-activated protein kinase (MAPK, 1K) family members ERK, JNK, and p38 play a divergent role in either promoting tumorigenesis or tumor-suppression. Activation of ERK and JNK promotes tumorigenesis; whereas, escalation of p38 inhibits carcinogenesis. As these three MAPK members are controlled by the common up-stream MAPK signaling proteins which consist of MAPK kinases (2K) and MAPK kinase kinases (3K), how to selectively actuate tumor-suppressive p38, not concurrently stimulate tumorigenic ERK and JNK, in cancer cells is a challenge for cancer researchers, and a new opportunity for novel anti-cancer drug discovery. Using human pancreatic cancer cells and xenograft mice as models, we found that a small molecule triptonide first discerningly activated the up-stream MAPK kinase kinase MEKK4, not the other two 3K members ASK1 and GADD45; and then selectively actuated the middle stream MAPK kinase MKK4, not the other two 2K members MKK3 and MKK6; and followed by activation of the MAPK member p38, not the other two members ERK and JNK. These data suggest that triptonide is a selective MEKK4-MKK4-p38 axis agonist. Consequently, selective activation of the MEKK4-MKK4-p38 signaling axis by triptonide activated tumor suppressor p21 and inhibited CDK3 expression, resulting in cancer cell cycle arrest at G2/M phase and marked inhibition of pancreatic cancer cell tumorigenic capability in vitro and tumor growth in xenograft mice. Our findings support the notion that selective activation of tumor-suppressive MEKK4-MKK4-p38-p21signaling pathway by triptonide is a new approach for pancreatic cancer therapy, providing a new drug candidate for development of novel anti-cancer therapeutics.

Luminescent ruthenium(II) polypyridyl complexes acted as radiosensitizer for pancreatic cancer by enhancing radiation-induced DNA damage.

Background: Pancreatic cancer is a highly lethal malignancy which ranks 4th most common cause of cancer death in US and 6th in China. Novel drugs are required to improve the survival and prognosis of patients.Methods: Ruthenium(II) complexes with variation number of DIP ligand were synthesized and further adopted as radiosensitizer for pancreatic cancer. The influence of ruthenium(II) complexes on cell behaviors and tumor growth were investigated. The DNA binding affinity of ruthenium(II) complexes and plasmid was measured by using agarose gel electrophoresis.Results: Luminescent ruthenium(II) complex can rapidly enter into cell nuclei and consequently combine with DNA, resulting in the enhanced DNA damage induced by X-ray irradiation. Upon intratumoral injection of ruthenium(II) complex, excellent tumor growth inhibition was accomplished under ionizing radiation of human pancreatic cancer xenograft nude mice.Conclusions: Taken together, our study suggest that the ruthenium(II) polypyridyl complexes can effectively enhance radiation-induced DNA damage, which is likely to benefit the imaging-guided cancer radio-chemotherapy.

Adiponectin Suppresses Human Pancreatic Cancer Growth through Attenuating the β-Catenin Signaling Pathway.

Adipokines are emerging as a link between obesity and obesity-related cancers, including pancreatic cancer. Adiponectin is an abundant adipokine with pleiotropic beneficial roles in metabolic disorders. Low adiponectin levels are commonly observed in human obesity and have been associated with increased pancreatic cancer risk in prospective epidemiologic studies. Here, we investigated the direct effect of adiponectin on human pancreatic cancer in vitro and in vivo. Our results showed that adiponectin treatment significantly inhibited the proliferation of human pancreatic cancer cells. Knockdown of adiponectin receptors completely eliminated the antiproliferation effect of adiponectin and markedly promoted the growth of human pancreatic cancer xenografts in nude mice. Further analysis revealed that adiponectin blocked the phosphorylation/inactivation of GSK-3β, suppressed the intracellular accumulation of β-catenin, reduced the expression of cyclin D1, and consequently caused cell cycle accumulation at the G0-G1 phase in pancreatic cancer cells. Adiponectin-mediated attenuation of cell proliferation was abrogated by the GSK-3β inhibitor. In addition, a microarray analysis revealed that adiponectin also downregulated the expression of TCF7L2, a coactivator of β-catenin, at the transcriptional level in pancreatic cancer cells. These results indicated that the protective role of adiponectin against human pancreatic cancer might be attributed to its attenuating effect on the β-catenin signaling pathway. Taken together, our findings support a causal link between hypoadiponectinemia and increased pancreatic cancer risk, and suggest that activating adiponectin signaling could be a novel therapeutic strategy for obesity-related pancreatic cancer.

Radioimmunotherapy of PANC-1 Human Pancreatic Cancer Xenografts in NRG Mice with Panitumumab Modified with Metal-Chelating Polymers Complexed to 177Lu.

Our aim was to evaluate the effectiveness and normal tissue toxicity of radioimmunotherapy (RIT) of s.c. PANC-1 human pancreatic cancer (PnCa) xenografts in NRG mice using anti-EGFR panitumumab linked to metal-chelating polymers (MCPs) that present 13 DOTA chelators to complex the β-emitter, 177Lu. The clonogenic survival (CS) of PANC-1 cells treated in vitro with panitumumab-MCP-177Lu (0.3-1.2 MBq) and DNA double-strand breaks (DSBs) in the nucleus of these cells were measured by confocal immunofluorescence microscopy for γ-H2AX. Subcellular distribution of radioactivity for panitumumab-MCP-177Lu was measured, and absorbed doses to the cell nucleus were calculated. Normal tissue toxicity was assessed in non tumor-bearing NRG mice by monitoring body weight, complete blood cell counts (CBC), serum alanine aminotransferase (ALT), and creatinine (Cr) after i.v. injection of 6 MBq (10 μg) of panitumumab-MCP-177Lu. RIT was performed in NRG mice with s.c. PANC-1 tumors injected i.v. with 6 MBq (10 μg) of panitumumab-MCP-177Lu. Control mice received nonspecific human IgG-MCP-177Lu (6 MBq; 10 μg), unlabeled panitumumab (10 μg), or normal saline. The tumor growth index (TGI) was compared. Tumor and normal organ doses were estimated based on biodistribution studies. Panitumumab-MCP-177Lu reduced the CS of PANC-1 cells in vitro by 7.7-fold at the highest amount tested (1.2 MBq). Unlabeled panitumumab had no effect on the CS of PANC-1 cells. γ-H2AX foci were increased by 3.8-fold by panitumumab-MCP-177Lu. Panitumumab-MCP-177Lu deposited 3.84 Gy in the nucleus of PANC-1 cells. Administration of panitumumab-MCP-177Lu (6 MBq; 10 μg) to NRG mice caused no change in body weight, CBC, or ALT and only a slight increase in Cr compared to NRG mice treated with normal saline. Panitumumab-MCP-177Lu strongly inhibited tumor growth in NRG mice (TGI = 2.3 ± 0.2) compared to normal saline-treated mice (TGI = 5.8 ± 0.5; P < 0.01). Unlabeled panitumumab had no effect on tumor growth (TGI = 6.0 ± 1.6; P > 0.05). The absorbed dose of PANC-1 tumors was 12.3 Gy. The highest normal organ doses were absorbed by the pancreas, liver, spleen, and kidneys. We conclude that EGFR-targeted RIT with panitumumab-MCP-177Lu was able to overcome resistance to panitumumab in KRAS mutant PANC-1 tumors in NRG mice and may be a promising approach to treatment of PnCa in humans.

Abstract 3482: Cachexia has profound metabolic consequences in the heart and skeletal muscle

Abstract Cachexia is an underexplored and yet devastating consequence of cancer that is the cause of 20% of all cancer related deaths [1]. Cachexia is associated with poor treatment outcome [3], fatigue, and extremely poor quality of life [2, 3]. Tissue wasting is one of the characteristics associated with cachexia syndrome. Here we have used 1H MRS to characterize the metabolic profile of skeletal muscle and heart muscle obtained from normal mice and noncachexia (Panc1) and cachexia inducing (Pa04C) human pancreatic cancer xenograft bearing mice to further understand this syndrome. Male severe combined immunodeficient (SCID) mice were inoculated in the right flank with cancer cells (2 × 106) and in the right hind leg muscle with cachexia reporter myoblasts (2×106) [4]. Mice were sacrificed once tumors were ~400 mm3, and the heart and skeletal muscle were harvested for dual phase solvent extraction. 1H magnetic resonance (MR) spectra of the water phase were acquired with a 750 MHz MR spectrometer. Quantitative changes in metabolite levels were obtained from 1H MR spectra. Leucine, creatine, lactate and glucose were significantly lower, and acetate and formate were significantly higher, in the muscle of cachectic mice compared to non-cachetic mice. Cachectic mice had significantly lower alanine, succinate, glycine, lipid and PUFA (poly unsaturated fatty acids), and significantly higher acetate, pyruvate, and formate compared to normal mice. Non-cachectic mice had significantly lower alanine, succinate, phosphocreatine, and glycine, and significantly higher leucine, isoleucine, valine, pyruvate, creatine, taurine and glucose compared to normal mice. In the heart, leucine, isoleucine, valine, aspartate, glucose, lipid and PUFA were significantly lower, and glutamine was significantly higher, in cachectic mice compared to non-cachetic mice. Cachectic mice had significantly lower leucine, isoleucine, valine, lactate, alanine, glutamate, and PUFA, and significantly higher glutamine and glucose compared to normal mice. Non-cachectic mice had significantly lower alanine, and significantly higher glucose and lipid compared to normal mice. These data highlight, for the first time, the profound metabolic changes that occur in skeletal muscle and the heart with cachexia, identifying potential in vivo 1H MRS indices to detect the onset of cachexia from changes in branched chain amino acids, glucose, and PUFAs. Our data also provide new insights into the effects of cachexia as well as noncachexia inducing tumors on skeletal muscle and heart metabolism that may lead to metabolic interventions in ‘metabolotheranostic' strategies to reduce the morbidity associated with cancer and cachexia. Supported by NIH R01CA193365, R35CA209960, and P30CA06973. Ref. 1. Argiles et al: Nat. Rev. Can 2014, 14(11):754 2. Fearon et al: HPB (Oxford) 2010, 12(5):323 3. Ozola et al: Pancreatology 2015, 15(1):19 4. Winnard et al: Can Res. 2016, 76(6):1441 Citation Format: Santosh Kumar Bharti, Paul Winnard, Yelena Mironchik, Marie-France Penet, Zaver M. Bhujwalla. Cachexia has profound metabolic consequences in the heart and skeletal muscle [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3482.

Extracellular Citrate Affects Critical Elements of Cancer Cell Metabolism and Supports Cancer Development In Vivo.

Glycolysis and fatty acid synthesis are highly active in cancer cells through cytosolic citrate metabolism, with intracellular citrate primarily derived from either glucose or glutamine via the tricarboxylic acid cycle. We show here that extracellular citrate is supplied to cancer cells through a plasma membrane-specific variant of the mitochondrial citrate transporter (pmCiC). Metabolomic analysis revealed that citrate uptake broadly affected cancer cell metabolism through citrate-dependent metabolic pathways. Treatment with gluconate specifically blocked pmCiC and decreased tumor growth in murine xenografts of human pancreatic cancer. This treatment altered metabolism within tumors, including fatty acid metabolism. High expression of pmCiC was associated with invasion and advanced tumor stage across many human cancers. These findings support the exploration of extracellular citrate transport as a novel potential target for cancer therapy.Significance: Uptake of extracellular citrate through pmCiC can be blocked with gluconate to reduce tumor growth and to alter metabolic characteristics of tumor tissue. Cancer Res; 78(10); 2513-23. ©2018 AACR.

Norleual, a hepatocyte growth factor and macrophage stimulating protein dual antagonist, increases pancreatic cancer sensitivity to gemcitabine.

Pancreatic cancer is a leading cause of cancer deaths in the USA and is characterized by an exceptionally poor long-term survival rate compared with other major cancers. The hepatocyte growth factor (HGF) and macrophage stimulating protein (MSP) growth factor systems are frequently over-activated in pancreatic cancer and significantly contribute to cancer progression, metastasis, and chemotherapeutic resistance. Small molecules homologous to the 'hinge' region of HGF, which participates in its dimerization and activation, had been developed and shown to bind HGF with high affinity, antagonize HGF's actions, and possess anticancer activity. Encouraged by sequence homology between HGF's hinge region and a similar sequence in MSP, our laboratory previously investigated and determined that these same antagonists could also block MSP-dependent cellular responses. Thus, the purpose of this study was to establish that the dual HGF/MSP antagonist Norleual could inhibit the prosurvival activity imparted by both HGF and MSP to pancreatic cancer cells in vitro, and to determine whether this effect translated into an improved chemotherapeutic impact for gemcitabine when delivered in combination in a human pancreatic cancer xenograft model. Our results demonstrate that Norleual does indeed suppress HGF's and MSP's prosurvival effects as well as sensitizing pancreatic cancer cells to gemcitabine in vitro. Most importantly, treatment with Norleual in combination with gemcitabine markedly inhibited in-vivo tumor growth beyond the suppression observed with gemcitabine alone. These results suggest that dual functional HGF/MSP antagonists like Norleual warrant further development and may offer an improved therapeutic outcome for pancreatic cancer patients.

Facile assembly of upconversion nanoparticle-based micelles for active targeted dual-mode imaging in pancreatic cancer

BackgroundPancreatic cancer remains the leading cause of cancer-related deaths, the existence of cancer stem cells and lack of highly efficient early detection may account for the poor survival rate. Gadolinium ion-doped upconversion nanoparticles (UCNPs) provide opportunities for combining fluorescent with magnetic resonance imaging, and they can improve the diagnostic efficacy of early pancreatic cancer. In addition, as one transmembrane glycoprotein overexpressed on the pancreatic cancer stem cells, CD326 may act as a promising target. In this study, we developed a facile strategy for developing anti-human CD326-grafted UCNPs-based micelles and performed the corresponding characterizations. After conducting in vitro and vivo toxicology experiments, we also examined the active targeting capability of the micelles upon dual-mode imaging in vivo.ResultsWe found that the micelles owned superior imaging properties and long-time stability based on multiple characterizations. By performing in vitro and vivo toxicology assay, the micelles had good biocompatibility. We observed more cellular uptake of the micelles with the help of anti-human CD326 grafted onto the micelles. Furthermore, we successfully concluded that CD326-conjugated micelles endowed promising active targeting ability by conducting dual-mode imaging in human pancreatic cancer xenograft mouse model.ConclusionsWith good biocompatibility and excellent imaging properties of the micelles, our results uncover efficient active homing of those micelles after intravenous injection, and undoubtedly demonstrate the as-obtained micelles holds great potential for early pancreatic cancer diagnosis in the future and would pave the way for the following biomedical applications.

Panitumumab Modified with Metal-Chelating Polymers (MCP) Complexed to 111In and 177Lu-An EGFR-Targeted Theranostic for Pancreatic Cancer.

A metal-chelating polymer (MCP) with a polyglutamide (PGlu) backbone presenting on average 13 DOTA (tetraazacyclododecane-1,4,7,10-tetraacetic acid) chelators for complexing 111In or 177Lu and 10 polyethylene glycol (PEG) chains to minimize liver and spleen uptake was conjugated to antiepidermal growth factor receptor (EGFR) monoclonal antibody (mAb), panitumumab. Because panitumumab-MCP may be dual-labeled with 111In and 177Lu for SPECT, or radioimmunotherapy (RIT) exploiting the Auger electrons or β-particle emissions, respectively, we propose that panitumumab-MCP could be a useful theranostic agent for EGFR-positive PnCa. Bioconjugation was achieved by reaction of a hydrazine nicotinamide (HyNIC) group on the MCP with an aryl aromatic aldehyde introduced into panitumumab by reaction with succinimidyl-4-formylbenzamide (S-4FB). The conjugation reaction was monitored by measurement of the chromophoric bis-aryl hydrazone bond formed (ε350nm = 24 500 M-1 cm-1) to achieve two MCPs/panitumumab. Labeling of panitumumab-MCP with 111In or 177Lu demonstrated that masses as small as 0.1 μg were labeled to >90% labeling efficiency (L.E.) and a specific activity (SA) of >70 MBq/μg. Panitumumab-DOTA incorporating two DOTA per mAb was labeled with 111In or 177Lu to a maximum SA of 65 MBq/μg and 46 MBq/μg, respectively. Panitumumab-MCP-177Lu exhibited saturable binding to EGFR-overexpressing MDA-MB-468 human breast cancer cells. The Kd for binding of panitumumab-MCP-177Lu to EGFR (2.2 ± 0.6 nmol/L) was not significantly different than panitumumab-DOTA-177Lu (1.0 ± 0.4 nmol/L). 111In and 177Lu were stably complexed to panitumumab-MCP. Panitumumab-MCP-111In exhibited similar whole body retention (55-60%) as panitumumab-DOTA-111In in NOD-scid mice up to 72 h postinjection (p.i.) and equivalent excretion of radioactivity into the urine and feces. The uptake of panitumumab-MCP-111In in most normal tissues in NOD-scid mice with EGFR-positive PANC-1 human pancreatic cancer (PnCa) xenografts at 72 h p.i. was not significantly different than panitumumab-DOTA-111In, except for the liver which was 3-fold greater for panitumumab-MCP-111In. Tumor uptake of panitumumab-MCP-111In (6.9 ± 1.3%ID/g) was not significantly different than panitumumab-DOTA-11In (6.6 ± 3.3%ID/g). Tumor uptake of panitumumab-MCP-111In and panitumumab-DOTA-111In were reduced by preadministration of excess panitumumab, suggesting EGFR-mediated uptake. Tumor uptake of nonspecific IgG-MCP (5.4 ± 0.3%ID/g) was unexpectedly similar to panitumumab-MCP-111In. An increased hydrodynamic radius of IgG when conjugated to an MCP may encourage tumor uptake via the enhanced permeability and retention (EPR) effect. Tumor uptake of panitumumab-DOTA-111In was 3.5-fold significantly higher than IgG-DOTA-111In. PANC-1 tumors were imaged by microSPECT/CT at 72 h p.i. of panitumumab-MCP-111In or panitumumab-DOTA-111In. Tumors were not visualized with preadministration of excess panitumumab to block EGFR, or with nonspecific IgG radioimmunoconjugates. We conclude that linking panitumumab to an MCP enabled higher SA labeling with 111In and 177Lu than DOTA-conjugated panitumumab, with preserved EGFR binding in vitro and comparable tumor localization in vivo in mice with s.c. PANC-1 human PnCa xenografts. Normal tissue distribution was similar except for the liver which was higher for the polymer radioimmunoconjugates.

MicroRNA-410-3p attenuates gemcitabine resistance in pancreatic ductal adenocarcinoma by inhibiting HMGB1-mediated autophagy.

Gemcitabine-based chemotherapy is the most common treatment option for pancreatic ductal adenocarcinoma (PDAC). However, it offers little therapeutic value in many cases due to the rapid development of chemoresistance. MicroRNAs (miRNAs) have been found to play pivotal roles in the chemotherapeutic resistance of PDAC. We found that miR-410-3p was significantly down-regulated in human pancreatic cancer xenograft (HPCx) tumor tissues from gemcitabine-treated mice. Low miR-410-3p expression correlated with gemcitabine resistance in HPCx tumors and PDAC cells as well as poor prognosis in PDAC patients. We also found that miR-410-3p attenuated the gemcitabine resistance of PDAC by targeting the 3′-UTR of HMGB1. Moreover, our study clearly demonstrated that miR-410-3p enhanced chemosensitivity to gemcitabine via inhibiting HMGB1-induced autophagy during chemotherapy in PDAC cells. Our study suggests that miR-410-3p expression may be a useful indicator of the potential for chemoresistance to gemcitabine and provide a potential new therapeutic target for chemoresistance in PDAC.

Novel TREM-1 Inhibitors Attenuate Tumor Growth and Prolong Survival in Experimental Pancreatic Cancer.

Pancreatic cancer (PC) is a highly lethal cancer with an urgent need to expand the limited treatment options for patients. Tumor-associated macrophages (TAMs) promote tumor aggressiveness and metastasis. High expression of triggering receptor expressed on myeloid cells 1 (TREM-1) on TAMs directly correlates with poor survival in patients with non-small cell lung cancer (NSCLC). We have previously hypothesized that blockade of TREM-1 could be a promising therapeutic strategy to treat cancer and shown that the novel, ligand-independent TREM-1 inhibitory peptides rationally designed using the signaling chain homooligomerization (SCHOOL) strategy suppress NSCLC growth in vivo. Here, we evaluated the therapeutic potential of these inhibitors in three human PC xenograft mouse models. Administration of SCHOOL peptides resulted in a strong antitumor effect achieving an optimal treatment/control (T/C) value of 19% depending on the xenograft and formulation used and persisting even after treatment was halted. The effect correlated significantly with increased survival and suppressed TAM infiltration. The peptides were well-tolerated when deployed either in free form or formulated into lipopeptide complexes for peptide half-life extension and targeted delivery. Finally, blockade of TREM-1 significantly reduced serum levels of interleukin (IL)-1α, IL-6, and macrophage colony-stimulating factor (M-CSF), but not vascular endothelial growth factor, suggesting M-CSF-dependent antitumor mechanisms. Collectively, these promising data suggest that SCHOOL TREM-1-specific peptide inhibitors have a cancer type independent, therapeutically beneficial antitumor activity and can be potentially used as a stand-alone therapy or as a component of combinational therapy for PC, NSCLC, and other solid tumors.

Histopathological and immunohystochemical study of human pancreatic cancer xenograft

Patients with pancreatic ductal adenocarcinoma and synchronous liver metastases (PACLM) have an extremely limited life expectancy. We performed a single-center analysis to explore the clinical results and prognostic factors of patients with PACLM receiving palliative care.We retrospectively reviewed 189 patients undergoing palliative care at Tianjin Medical University Cancer Hospital over a 15-year period. Clinical characteristics, survival condition, and factors associated with survival were analyzed. Treatment methods included palliative bypass surgery, percutaneous transhepatic cholangiodrainage, drug analgesia, symptomatic treatment, and other nutritional or supportive measures.The overall survival (OS) was 3.6 months for all patients. Multivariate analysis for clinical features showed that Karnofsky performance score (KPS), ascites, cigarette smoking, primary tumor size, and lactate dehydrogenase (LDH) were prognostic variables with statistical significance (P < 0.05). The patients were classified into three groups of patients according to how many of these 5 risk factors were present: 0–1, 2, or 3–5 risk factors. The median OS of the 3 groups of patients were 5.0, 3.3, and 2.5 months, respectively, with a notable statistical significance (P < 0.0001).KPS<80, ascites, cigarette smoking, primary tumor size≥5 cm, and LDH≥250U/L are effective predictive factors of poor prognosis for patients with PACLM. The stratification of treatment outcome groups based on these factors facilitates evaluation of individual prognosis and can guide clinical decisions.