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Triple Negative Breast Cancer (TNBC) is an aggressive subtype of breast cancer with high metastatic potential and increased morbidity and mortality. Although expression of, and signaling by the epidermal growth factor receptor (EGFR) is commonly seen in TNBC, anti-EGFR antibodies such as Cetuximab have had limited therapeutic efficacy, used alone or in combination with chemotherapy. Primary TNBC tumor growth and metastases require supporting vasculature, which develops through a combination of endothelial angiogenesis and vasculogenic mimicry (VM). VM is more frequently seen in TNBC than other breast cancer subtypes, and is associated with aggressive metastatic behavior. We previously developed αEGFR-E-P125A, an antibody-endostatin fusion protein, linking an anti-EGFR antibody targeting domain to a mutated version of the anti-angiogenic protein endostatin (E-P125A). αEGFR-E-P125A delivers a dimeric E-P125A payload which inhibits TNBC angiogenesis and VM in vitro and in vivo, and markedly decreases metastasis in both the MDA-MB-231-4175 and MDA-MB-468 TNBC xenograft models. To determine the mechanism of αEGFR-E-P125A action on inhibition of VM, we studied the effects of αEGFR-E-P125A on both the transcriptome and proteome. RNA-seq was conducted on MDA-MB-231-4175 TNBC cells plated on matrigel and undergoing VM, and on TNBC cells plated in matrigel and treated with αEGFR-E-P125A. Gene set enrichment analysis demonstrated that αEGFR-E-P125A treatment downregulated genes on the KRAS, JAK-STAT, and angiogenesis signaling pathways, including EGF, VEGFA, STAT3, PTK2, ITGB1, ITGB3, ITGAV, and ITGA5. Phospho-array analysis was used to interrogate the proteome and demonstrated downregulation of phosphorylation on multiple sites downstream of the EGFR and the α5β1 integrin receptors, such as the EGFR Y1069 site, the FAK Y397 site, implicated in the regulation of endothelial and TNBC motility, and the STAT3 Y705 and S727 sites, known for their role in promoting the transcription of angiogenic genes. Since inhibition of EGFR signaling alone does not inhibit VM, we interrogated the mechanistic relationship between αEGFR-E-P125A and VM inhibition through the α5β1 integrin/FAK signaling pathway. Specific inhibition of FAK at the Y397 site using the small molecule inhibitor, PF-573228 inhibited TNBC VM in vitro. Transient siRNA knockdown of FAK in MDA-MB-231-4175 cells and shRNA-mediated knockdown of FAK in MDA-MB-231 TNBC cells confirmed that downregulation of FAK inhibited VM in vitro. To understand the effect of αEGFR-E-P125A on the EGFR and α5β1 integrin receptors, competition assays were conducted. αEGFR-E-P125A competed with EGF for the EGFR receptor and with the binding of fibronectin to α5β1 integrin. Treatment of TNBC cells with αEGFR-E-P125A reduced total α5 integrin protein levels and decreased co-localization of EGFR and α5β1 integrin receptors. These results indicate that αEGFR-E-P125A is bound to both EGFR and α5β1 integrin, simultaneously suppressing downstream EGFR and integrin signaling. Simultaneous inhibition of EGFR and α5β1integrin signaling by αEGFR-E-P125A fusion is a promising approach to inhibition of TNBC growth and metastases. Citation Format: Ankita P. Sankar, Hava Gil Henn, Hyun Mi Cho, Dania Nassar, Sundaram Ramakrishnan, Rathin Das, Yu Zhang, Christian Elledge, Seung-Uon Shin, Joseph Rosenblatt. αEGFR-E-P125A ANTIBODY-ENDOSTATIN FUSION PROTEIN REDUCES VASCULOGENIC MIMICRY AND TUMOR CELL MOTILITY BY INHIBITION OF EGFR, INTEGRIN AND FAK SIGNALING [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-24-06.

Opioids are the gold standard for chronic and acute pain management; however, their consequence on gastric function is relatively understudied. Opioid users have a higher incidence of gastric dysfunction, worse quality of life, increased hospitalizations, and increased use of antiemetic and pain modulator medications. The current study shows that morphine treatment in the murine model results in greater disruption of gastric epithelial cell morphology, increased gastric cell apoptosis, elevated inflammatory cytokines, and matrix metallopeptidase-9 secretion. Morphine treatment also increases gastric acid secretion and causes delays in gastric emptying. Moreover, morphine treatment causes an increase in systemic IL-6 level, which plays an important role in morphine-induced delayed gastric emptying and gastric damage. IL-6 knockout mice show a significant level of reduction in morphine-induced gastric delaying, acid retention, and gastric damage. Thus, morphine-mediated gastric damage is a consequence of the accumulation of acid in the stomach due to increased gastric acid secretion and delayed gastric emptying. Treatment with a proton pump inhibitor resulted in a significant reduction in morphine-induced gastric inflammation, gastric delaying, and improved morphine tolerance. Hence, these studies attribute morphine-mediated induction in gastric acidity and inflammatory cytokines as drivers for morphine-associated gastric pathology and show the therapeutic use of proton pump inhibitors as an inexpensive approach for clinical management of morphine-associated pathophysiology and analgesic tolerance.

Genes upregulated in KPC tumors vs mouse PDC

Quantitation of area stained in immunohistochenistry

Purpose: Pancreatic cancer stromal microenvironment is considered to be the major reason for failure of conventional and targeted therapy for this disease. The desmoplastic stroma, comprising mainly collagen and glycosaminoglycans like hyaluronan (HA), is responsible for compression of vasculature in the tumor resulting in impaired drug delivery and poor prognosis. Minnelide, a water-soluble prodrug of triptolide currently in phase I clinical trial, has been very effective in multiple animal models of pancreatic cancer. However, whether Minnelide will have efficacious delivery into the tumor despite the desmoplastic stroma has not been evaluated before.Experiment Design: Patient tumor-derived xenografts (PDX) and spontaneous pancreatic cancer mice were treated with 0.42 and 0.21 mg/kg body weight for 30 days. Stromal components were determined by IHC and ELISA-based assays. Vascular functionality and drug delivery to the tumor were assessed following treatment with Minnelide.Result: Our current study shows that treatment with Minnelide resulted in reduction of ECM components like HA and collagen in the pancreatic cancer stroma of both the spontaneous KPC mice as well as in patient tumor xenografts. Furthermore, treatment with Minnelide improved functional vasculature in the tumors resulting in four times more functional vessels in the treated animals compared with untreated animals. Consistent with this observation, Minnelide also resulted in increased drug delivery into the tumor compared with untreated animals. Along with this, Minnelide also decreased viability of the stromal cells along with the tumor cells in pancreatic adenocarcinoma.Conclusions: In conclusion, these results are extremely promising as they indicate that Minnelide, along with having anticancer effects is also able to deplete stroma in pancreatic tumors, which makes it an effective therapy for pancreatic cancer. Clin Cancer Res; 22(2); 415–25. ©2015 AACR.

Expression and activity of HA synthesis and degradation genes

Gene expression of collagen stabilization genes

Gene expression in normal ductal cells and cancer cells

IC50 of mouse CAFs and PDACs after 48h treatment with triptolide in vitro.

Opioids are commonly used for the management of cancer-associated pain and chemotherapy-induced diarrhea. The chemotherapeutic agent CPT-11 causes severe GI toxicity as a result of deconjugation of its inactive metabolite SN-38G by intestinal bacterial β-glucuronidases to the pharmacologically active SN-38. As opioids are known to cause gut microbial dysbiosis, this study evaluated whether CPT-11's anti-tumor efficacy and GI toxicity are exacerbated by opioid co-administration.8-week-old C57BL/6 male mice were treated with CPT-11 ± opioid co-administration. 16S rRNA sequencing was used for gut microbiome analysis. LC-MS analyses of plasma and intestinal extracts were performed to investigate the pharmacokinetic profile of CPT-11. Histological analysis and qRT-PCR were used to determine the severity of intestinal tissue damage. The in vitro assay using human liver microsome was also performed to confirm the effects of opioids on CPT-11 metabolism.Gut microbiome analysis showed that morphine treatment induced enrichment of β-glucuronidase-producing bacteria in the intestines of CPT-11-treated mice, resulting in SN-38 accumulation and exacerbation of GI toxicity in the small intestine. Oral administration of both antibiotics and a glucuronidase inhibitor protected mice against GI toxicity induced with CPT-11 and morphine co-administration, implicating a microbiome-dependent mechanism. Additionally, morphine and loperamide decreased the plasma concentration of SN-38 and compromised CPT-11's anti-tumor efficacy, although this seemed to be microbiome-independent.Gut microbiota play a significant role in opioid and chemotherapeutic agent drug-drug interactions. Inhibition of gut microbial glucuronidase may also prevent adverse GI effects of CPT-11 in patients on opioids.

Extracellular vesicles (EVs) are effective mediators of intercellular communications between enterocytes and immune cells. The current study showed that EVs isolated from mouse and human intestinal organoids modulated inflammatory responses of various immune cells including mouse bone-marrow derived-macrophages, dendritic cells, microglia cells, and human monocytes. EVs suppressed LPS-elicited cytokine production in these cells while morphine abolished EVs' immune modulatory effects. Microarray analysis showed that various microRNAs, especially Let-7, contributed to EV-mediated immune modulation. Using murine models, we showed that injection of EVs derived from intestinal organoids reduced endotoxin-induced systemic inflammation and alleviated the symptoms of DSS-induced colitis. EVs derived from morphine-treated organoids failed to suppress the immune response in both these models. Our study suggests that EVs derived from intestinal crypt cells play crucial roles in maintaining host homeostasis and opioid use is a risk factor for exacerbating inflammation in patients with inflammatory diseases such as sepsis and colitis.

BACKGROUND RHD is the leading cause of morbidity and mortality in the underdeveloped world. The burden of RHD has been estimated variably depending on the method and tool of examination. Recent echocardiography-based studies, generally accepted as the best tools to detect RHD, had shown 10-fold increased prevalence of RHD compared to clinical examination albeit fear for overestimation of the disease. While studies using more stringent criteria of WHF 2012 are limited and no such study has been conducted in this part of the country, we felt it necessary to conduct such a study in this hilly state of India. MATERIALS AND METHODS This is a community based cross sectional study in which each and every selected child aged 5-15 years from randomly selected schools of Manipur were examined physically and by 2D colour Doppler Echocardiography. The anthropometric parameters, clinical details and echocardiography findings were all recorded. Echocardiography loops were recorded for review by another cardiologist later. Analysis was done by using T test, descriptive statistics and with 95% confidence interval. All the analysis was done using STATA 13.0 (Stata Corp, USA). RESULTS 3600 children were screened in two years. The mean age was 11.77 years ± 2.50 SD, 1865 (51.8%) were male, 1442 (40%) were from government school and majority belonged to rural population (67%). Only 1 case of Clinical RHD with a combination of MR and AS was found giving a prevalence of 0.28/1000 (CI: 0.04-1.97). Echocardiography detected 3 cases of definite subclinical (prevalence rate of 0.83/1000 (CI: 0.27-2.58) and 14 cases of borderline subclinical RHD (prevalence rate of 3.9/1000 (CI: 2.30-6.56) befitting WHF 2012 criteria. CONCLUSION Prevalence of clinical RHD (0.28/1000) is very low compared to that of other Indian states. Using echocardiography, the prevalence becomes several folds higher compared to clinical examination alone, 0.83/1000 for Definite Subclinical and 3.9/1000 for Borderline Subclinical RHD. Further follow up studies using less stringent criteria (modified WHO criteria), may still have a role in detection of true burden of RHD in our community from the public health point of view.

Heat shock protein 70 (Hsp70), a protein chaperone, is known to promote cell survival and tumor progression. However, its role in the tumor microenvironment (TME) is largely unknown. We specifically evaluated Hsp70 in the TME by implanting tumors in wild-type (WT) controls or Hsp70−/- animals, thus creating a TME with or without Hsp70. Loss of Hsp70 led to significantly smaller tumors; there were no differences in stromal markers, but interestingly, depletion of CD8 + T-cells abrogated this tumor suppressive effect, indicating that loss of Hsp70 in the TME affects tumor growth through the immune cells. Compared to WT, adoptive transfer of Hsp70−/- splenocytes exhibited greater antitumor activity in immunodeficient NSG and Rag 1−/- mice. Hsp70−/- dendritic cells showed increased expression of MHCII and TNF-α both in vitro and in vivo. These results suggest that the absence of Hsp70 in the TME inhibits tumors through increased dendritic cell activation. Hsp70 inhibition in DCs may emerge as a novel therapeutic strategy against pancreatic cancer.

Prolonged exposure to opioids results in analgesic tolerance, drug overdose, and death. The mechanism underlying morphine analgesic tolerance still remains unresolved. We show that morphine analgesic tolerance was significantly attenuated in germfree (GF) and in pan-antibiotic−treated mice. Reconstitution of GF mice with naïve fecal microbiota reinstated morphine analgesic tolerance. We further demonstrated that tolerance was associated with microbial dysbiosis with selective depletion in Bifidobacteria and Lactobacillaeae. Probiotics, enriched with these bacterial communities, attenuated analgesic tolerance in morphine-treated mice. These results suggest that probiotic therapy during morphine administration may be a promising, safe, and inexpensive treatment to prolong morphine’s efficacy and attenuate analgesic tolerance. We hypothesize a vicious cycle of chronic morphine tolerance: morphine-induced gut dysbiosis leads to gut barrier disruption and bacterial translocation, initiating local gut inflammation through TLR2/4 activation, resulting in the activation of proinflammatory cytokines, which drives morphine tolerance.

Background: We have previously showed that gut microbiota-depleting oral antibiotics decrease tumor growth in C57BL/6J mice via the immune system (Sethi et al, Gastroenterology, 2018). The gut microbiota, however, includes thousands of species producing numerous metabolites. The search for individual bacterial species and metabolites that can promote cancer continues. Methods: We studied the effects of common microbiota modulation techniques in C57BL/6 mice procured from various commercial vendors and injected subcutaneously with cancer cells. 16S rRNA gene sequencing and metabolomics analyses were used to identify the putative cancer-promoting species and metabolites. Results: Mice procured from The Jackson Laboratory (JAX mice) form larger tumors at baseline compared to mice procured from the Charles River Laboratories (CR mice). JAX mice have tumor promoting microbiota that can be targeted by oral antibiotics unlike CR mice. This tumor-promoting microbiota gets transferred from JAX to CR mice via cohousing or fecal microbiota transplant. In a preclinical trial, oral vancomycin slowed the growth of cancer in avatar mice gavaged with stools of some pancreatic cancer patients but not of others. Statistical analyses reveal that the gut of JAX mice hosts a common human gram-positive gut commensal Ruminococcus gnavus that is associated with increased cancer growth in those mice. Monocolonization of germ-free mice with R gnavus directly increases tumor growth. Similarly, feeding R gnavus, but not a control bacterial species, brings the baseline tumor kinetics of CR mice up to that of JAX mice. Our experiments reveal that R gnavus needs sufficient dietary tryptophan (Trp) to promote tumor growth and in turn, catabolizes Trp into various metabolites including the Aryl Hydrocarbon Receptor (AHR) ligand Tryptamine, which we find, is also tumor-promoting per se. The tumor-modulating effects of oral vancomycin, tryptophan catabolites and R gnavus are abolished when murine AHR is stimulated by an exogenous ligand or when it is knocked out. Finally, we find that Ly6G+ cells are essential in effecting these bacteria-cancer interactions. Conclusions: We present initial evidence on how a common gut commensal hijacks an essential dietary amino acid to promote cancer via the AHR. We also demonstrate the essential but underappreciated role that the source of animal procurement plays in studies involving cancer models. Citation Format: Vrishketan Sethi, Junyi Tao, Saba Kurtom, Utpreksha Vaish, Farrukh Afaq, Shuan Zao, Xian Luo, Tejeshwar Jain, Prateek Sharma, Ejas Bava, Sundaram Ramakrishnan, Liang Li, Ashok Saluja, Vikas Dudeja. A common gut commensal utilizes tryptophan to promote cancer via the aryl hydrocarbon receptor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3046.

BackgroundThe role of lung epithelial cells in HIV-1-related lung comorbidities remains unclear, and the major hurdle in curing HIV is the persistence of latent HIV reservoirs in people living with HIV (PLWH). The advent of combined antiretroviral therapy has considerably increased the life span; however, the incidence of chronic lung diseases is significantly higher among PLWH. Lung epithelial cells orchestrate the respiratory immune responses and whether these cells are productively infected by HIV-1 is debatable.MethodsNormal human bronchial epithelial cells (NHBEs) grown on air–liquid interface were infected with X4-tropic HIV-1LAV and examined for latency using latency-reversing agents (LRAs). The role of CD4 and CXCR4 HIV coreceptors in NHBEs were tested, and DNA sequencing analysis was used to analyze the genomic integration of HIV proviral genes, Alu-HIVgag-pol, HIV-nef, and HIV-LTR. Lung epithelial sections from HIV-infected humans and SHIV-infected macaques were analyzed by FISH for HIV-gag-pol RNA and epithelial cell-specific immunostaining.Results and DiscussionNHBEs express CD4 and CXCR4 at higher levels than A549 cells. NHBEs are infected with HIV-1 basolaterally, but not apically, by X4-tropic HIV-1LAV in a CXCR4/CD4-dependent manner leading to HIV-p24 antigen production; however, NHBEs are induced to express CCR5 by IL-13 treatment. In the presence of cART, HIV-1 induces latency and integration of HIV provirus in the cellular DNA, which is rescued by the LRAs (endotoxin/vorinostat). Furthermore, lung epithelial cells from HIV-infected humans and SHIV-infected macaques contain HIV-specific RNA transcripts. Thus, lung epithelial cells are targeted by HIV-1 and could serve as potential HIV reservoirs that may contribute to the respiratory comorbidities in PLWH.

Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. Metastasis is the major cause of TNBC mortality. Angiogenesis facilitates TNBC metastases. Many TNBCs also form vascular channels lined by tumor cells rather than endothelial cells, known as ‘vasculogenic mimicry’ (VM). VM has been linked to metastatic TNBC behavior and resistance to anti-angiogenic agents. Epidermal growth factor receptor (EGFR) is frequently expressed on TNBC, but anti-EGFR antibodies have limited efficacy. We synthesized an anti-EGFR antibody–endostatin fusion protein, αEGFR IgG1-huEndo-P125A (αEGFR-E-P125A), designed to deliver a mutant endostatin, huEndo-P125A (E-P125A), to EGFR expressing tumors, and tested its effects on angiogenesis, TNBC VM, and motility in vitro, and on the growth and metastasis of two independent human TNBC xenograft models in vivo. αEGFR-E-P125A completely inhibited the ability of human umbilical vein endothelial cells to form capillary-like structures (CLS) and of TNBC cells to engage in VM and form tubes in vitro. αEGFR-E-P125A treatment reduced endothelial and TNBC motility in vitro more effectively than E-P125A or cetuximab, delivered alone or in combination. Treatment of TNBC with αEGFR-E-P125A was associated with a reduction in cytoplasmic and nuclear β-catenin and reduced phosphorylation of vimentin. αEGFR-E-P125A treatment of TNBC xenografts in vivo inhibited angiogenesis and VM, reduced primary tumor growth and lung metastasis of orthotopically implanted MDA-MB-468 TNBC cells, and markedly decreased lung metastases following intravenous injection of MDA-MB-231-4175 lung-tropic TNBC cells. Combined inhibition of angiogenesis, VM, and TNBC motility mediated by αEGFR-E-P125A is a promising strategy for the prevention of TNBC metastases.

SUMMARY Despite wide use of anti-vascular endothelial growth factor (VEGF) therapy for many solid cancers, most individuals become resistant to this therapy, leading to disease progression. Therefore, new biomarkers and strategies for blocking adaptive resistance of cancer to anti-VEGF therapy are needed. As described here, we demonstrate that cancer-derived small extracellular vesicles package increasing quantities of VEGF and other factors in response to anti-VEGF therapy. The packaging process of VEGF into small extracellular vesicles (EVs) is mediated by the tetraspanin CD63. Furthermore, small EV-VEGF (eVEGF) is not accessible to anti-VEGF antibodies and can trigger intracrine VEGF signaling in endothelial cells. eVEGF promotes angiogenesis and enhances tumor growth despite bevacizumab treatment. These data demonstrate a mechanism where VEGF is partitioned into small EVs and promotes tumor angiogenesis and progression. These findings have clinical implications for biomarkers and therapeutic strategies for ovarian cancer., Graphical abstract, In brief Ma et.al report that cancer-cell-derived small EVs contain increasing amounts of VEGF (eVEGF) and contribute to resistance to anti-VEGF therapy (AVT). CD63 is a potential mediator that regulates packaging of VEGF into small EVs. eVEGF can trigger intracrine VEGF signaling in endothelial cells and promote angiogenesis despite AVT.

Background and Aims Opioids are the most prescribed analgesics for pain in inflammatory bowel diseases [IBD]; however, the consequences of opioid use on IBD severity are not well defined. This is the first study investigating consequences of hydromorphone in both dextran sodium sulphate [DSS]-induced colitis and spontaneous colitis (IL-10 knockout [IL-10-/-]) mouse models of IBD. Methods To determine the consequences of opioids on IBD pathogenesis, wild-type [WT] mice were treated with clinically relevant doses of hydromorphone and colitis was induced via 3% DSS in drinking water for 5 days. In parallel we also determined the consequences of opioids in a spontaneous colitis model. Results Hydromorphone and DSS independently induced barrier dysfunction, bacterial translocation, disruption of tight junction organisation and increased intestinal and systemic inflammation, which were exacerbated in mice receiving hydromorphone in combination with DSS. Hydromorphone + DSS-treated mice exhibited significant microbial dysbiosis. Predictive metagenomic analysis of the gut microbiota revealed high abundance in the bacterial communities associated with virulence, antibiotic resistance, toxin production, and inflammatory properties. Hydromorphone modulates tight junction organisation in a myosin light chain kinase [MLCK]-dependent manner. Treatment with MLCK inhibitor ML-7 ameliorates the detrimental effects of hydromorphone on DSS-induced colitis and thus decreases severity of IBD. Similarly, we demonstrated that hydromorphone treatment in IL-10-/- mice resulted in accelerated clinical manifestations of colitis compared with control mice. Conclusions Opioids used for pain management in IBD accelerate IBD progression by dysregulation of the gut microbiota, leading to expansion of pathogenic bacteria, translocation of bacteria, immune deregulation and sustained inflammation.

BACKGROUND: Even after surgical resection, most patients with localized pancreatic ductal adenocarcinoma (PDAC) succumb to disease recurrence. Current animal models do not recapitulate this pattern of disease recurrence. Our goal was to develop a clinically relevant, immunocompetent model of PDAC resection to study recurrence and evaluate therapy. METHODS: Pancreatic cancer cells derived from tumors arising in KPC (LSL-Kras(G12D/+;) LSL-Trp53(R172H/+;) Pdx-1-Cre) mice were co-injected with stromal cells (pancreatic stellate cells) into the pancreas of immunocompetent mice to simulate the stroma rich tumors seen in human PDAC. After allowing tumors to form, we resected these localized tumors and followed the mice for tumor recurrence. Circulating tumor cells (CTCs) were isolated, and systemic chemotherapy or immunotherapy was administered following tumor resection. RESULTS: Tumors formed by co-injection of KPC cells and stromal cells demonstrated a dense desmoplastic reaction similar to that seen in human disease. Resection at days 15 and 21 after implantation revealed uniform tumor volumes of 92 ± 19 mm(3) on day 15 and 444 ± 54 mm(3) on Day 21. Histology of resected tumors showed negative margins. Resembling human PDAC, mice that underwent resection showed improved median survival (58 vs 47 days) but most animals developed intra-abdominal recurrence on follow up. Adjuvant chemotherapy (Median survival 69 vs 58 days), but not immunotherapy (Median Survival: 69 vs 65 days) tended towards improved survival as seen in human disease. Circulating-Tumor-Cells were reliably identified from mice with and without resection, suggesting utility of this model in studying tumor metastases and recurrence. CONCLUSION: We describe an immunocompetent animal model that recapitulates human disease in morphology and recurrence patterns. We show that it can be used to evaluate therapy in clinical scenarios associated with surgical resection and may help characterize factors responsible for disease recurrence.

// Devika Kir 1, 2 , Manju Saluja 1 , Shrey Modi 2 , Annapoorna Venkatachalam 1 , Erica Schnettler 1 , Sabita Roy 1, 2, 3 , Sundaram Ramakrishnan 1, 3 1 Department of Pharmacology, Masonic Comprehensive Cancer Center, Minneapolis, MN 55455, USA 2 Department of Surgery, University of Minnesota, Minneapolis, MN 55455, USA 3 Present address: Department of Surgery, University of Miami, FL 33136, USA Correspondence to: Sundaram Ramakrishnan, email: sunda001@umn.edu Keywords: cell-permeable iron, ferric ammonium citrate, angiogenesis, vascular endothelial growth factor, receptor phosphorylation Received: March 25, 2016 Accepted: July 19, 2016 Published: August 30, 2016 ABSTRACT Angiogenesis is important for tumor growth and metastasis. Hypoxia in tumors drives this angiogenic response by stabilizing Hypoxia Inducible Factors (HIF) and target genes like Vascular Endothelial Growth Factor (VEGF). HIF stability is regulated by Prolylhydroxylases (PHD)-mediated modification. Iron is an important cofactor in regulating the enzymatic activity of PHDs. Reducing intracellular iron, for instance, mimics hypoxia and induces a pro-angiogenic response. It is hypothesized that increasing the intracellular iron levels will have an opposite, anti-angiogenic effect. We tested this hypothesis by perturbing iron homeostasis in endothelial cells using a unique form of iron, Ferric Ammonium Citrate (FAC). FAC is a cell-permeable form of iron, which can passively enter into cells bypassing the transferrin receptor mediated uptake of transferrin-bound iron. Our studies show that FAC does not decrease the levels of HIF-1α and HIF-2α in endothelial cells but inhibits the autocrine stimulation of VEGF-Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) system by blocking receptor tyrosine kinase phosphorylation. FAC inhibits VEGF-induced endothelial cell proliferation, migration, tube formation and sprouting. Finally, systemic administration of FAC inhibits VEGF and tumor cell-induced angiogenesis in vivo . In conclusion, our studies show that cell-permeable iron attenuates VEGFR-2 mediated signaling and inhibits tumor angiogenesis.

Purpose: Pancreatic cancer stromal microenvironment is considered to be the major reason for failure of conventional and targeted therapy for this disease. The desmoplastic stroma, comprising mainly collagen and glycosaminoglycans like hyaluronan (HA), is responsible for compression of vasculature in the tumor resulting in impaired drug delivery and poor prognosis. Minnelide, a water-soluble prodrug of triptolide currently in phase I clinical trial, has been very effective in multiple animal models of pancreatic cancer. However, whether Minnelide will have efficacious delivery into the tumor despite the desmoplastic stroma has not been evaluated before. Experiment Design: Patient tumor-derived xenografts (PDX) and spontaneous pancreatic cancer mice were treated with 0.42 and 0.21 mg/kg body weight for 30 days. Stromal components were determined by IHC and ELISA-based assays. Vascular functionality and drug delivery to the tumor were assessed following treatment with Minnelide. Result: Our current study shows that treatment with Minnelide resulted in reduction of ECM components like HA and collagen in the pancreatic cancer stroma of both the spontaneous KPC mice as well as in patient tumor xenografts. Furthermore, treatment with Minnelide improved functional vasculature in the tumors resulting in four times more functional vessels in the treated animals compared with untreated animals. Consistent with this observation, Minnelide also resulted in increased drug delivery into the tumor compared with untreated animals. Along with this, Minnelide also decreased viability of the stromal cells along with the tumor cells in pancreatic adenocarcinoma. Conclusions: In conclusion, these results are extremely promising as they indicate that Minnelide, along with having anticancer effects is also able to deplete stroma in pancreatic tumors, which makes it an effective therapy for pancreatic cancer. Clin Cancer Res; 22(2); 415–25. ©2015 AACR.

Background & Aims Immunotherapies are ineffective against pancreatic cancer. We investigated whether the activity of nuclear factor (NF)κB in pancreatic stromal cells contributes to an environment that suppresses antitumor immune response. Methods Pancreata of C57BL/6 or Rag1–/– mice were given pancreatic injections of a combination of KrasG12D/+; Trp53 R172H/+; Pdx-1cre (KPC) pancreatic cancer cells and pancreatic stellate cells (PSCs) extracted from C57BL/6 (control) or mice with disruption of the gene encoding the NFκB p50 subunit (Nfkb1 or p50–/– mice). Tumor growth was measured as an endpoint. Other mice were given injections of Lewis lung carcinoma (LLC) lung cancer cells or B16-F10 melanoma cells with control or p50–/– fibroblasts. Cytotoxic T cells were depleted from C57BL/6 mice by administration of antibodies against CD8 (anti-CD8), and growth of tumors from KPC cells, with or without control or p50–/– PSCs, was measured. Some mice were given an inhibitor of CXCL12 (AMD3100) and tumor growth was measured. T-cell migration toward cancer cells was measured using the Boyden chamber assay. Results C57BL/6 mice coinjected with KPC cells (or LLC or B16-F10 cells) and p50–/– PSCs developed smaller tumors than mice given injections of the cancer cells along with control PSCs. Tumors that formed when KPC cells were injected along with p50–/– PSCs had increased infiltration by activated cytotoxic T cells along with decreased levels of CXCL12, compared with tumors grown from KPC cells injected along with control PSCs. KPC cells, when coinjected with control or p50–/– PSCs, developed the same-size tumors when CD8+ T cells were depleted from C57BL/6 mice or in Rag1–/– mice. The CXCL12 inhibitor slowed tumor growth and increased tumor infiltration by cytotoxic T cells. In vitro expression of p50 by PSCs reduced T-cell migration toward and killing of cancer cells. When cultured with cancer cells, control PSCs expressed 10-fold higher levels of CXCL12 than p50–/– PSCs. The CXCL12 inhibitor increased migration of T cells toward KPC cells in culture. Conclusions In studies of mice and cell lines, we found that NFκB activity in PSCs promotes tumor growth by increasing expression of CXCL12, which prevents cytotoxic T cells from infiltrating the tumor and killing cancer cells. Strategies to block CXCL12 in pancreatic tumor cells might increase antitumor immunity.