Inoculation Of Cancer Cells Research Articles

Establishment and Characterization of FusionRed Stable Transfected Canine Prostate Adenocarcinoma and Transitional Cell Carcinoma Cells.

Cancer cell inoculation is routinely used to evaluate novel therapeutic approaches in vivo. However, without reporter genes enabling deep tissue imaging, study of early tumor progression and therapeutic responses is often limited. We describe the establishment and characterization of two canine cancer cell lines stably expressing red fluorescence proteins as tools for later in vivo imaging. Two red fluorescence cell lines were generated by plasmid transfection. Fluorescence protein expression was confirmed by flow cytometry and microscopy. Deep tissue imaging was demonstrated in mice using a NightOWL LB 983. Gene expression changes after transfection were analyzed by RNAseq. Both cell lines were detectable in vivo by subcutaneous injection of 1×106 cells. RNAseq revealed up to 2005 transfection-induced differentially expressed genes but no significant changes in cellular key pathways. The fluorescent cell lines provide a solid basis for future in vivo studies on canine cancer.

Linoleic Acid Upregulates Microrna-494 to Induce Quiescence in Colorectal Cancer.

Cancer dormancy is a state characterized by the quiescence of disseminated cancer cells, and tumor recurrence occurs when such cells re-proliferate after a long incubation period. These cancer cells tend to be treatment resistant and one of the barriers to successful therapeutic intervention. We have previously reported that long-term treatment of cancer cells with linoleic acid (LA) induces a dormancy-like phenotype. However, the mechanism underpinning this effect has not yet been clarified. Here, we investigate the mechanism of LA-induced quiescence in cancer cells. We first confirmed that long-term treatment of the mouse colorectal cancer cell line CT26 with LA induced quiescence. When these cells were inoculated subcutaneously into a syngeneic mouse and fed with an LA diet, the inoculated cancer cells maintained the quiescent state and exhibited markers of dormancy. LA-treated CT26 cells showed reduced oxidative phosphorylation, glycolysis, and energy production as well as reduced expression of the regulatory factors Pgc1α and MycC. MicroRNA expression profiling revealed that LA induced an upregulation in miR-494. The expression of Pgc1α and MycC were both induced by an miR-494 mimic, and the LA-induced decrease in gene expression was abrogated by an miR-494 inhibitor. The expression of miR-494 was enhanced by the mitochondrial oxidative stress produced by LA. In a syngeneic mouse subcutaneous tumor model, growth suppression by an LA diet and growth delay by LA pretreatment + LA diet were found to have similar effects as administration of an miR-494 mimic. In contrast, the effects of LA were abrogated by an miR-494 inhibitor. Analysis of human colorectal cancer tissue revealed that miR-494 was present at low levels in non-metastatic cases and cases with simultaneous liver metastases but was expressed at high levels in cases with delayed liver metastases, which also exhibited reduced expression of PGC1α and MYCC. These results suggest that miR-494 is involved in cancer dormancy induced by high levels of LA intake and that this microRNA may be valuable in targeting dormant cancer cells.

Xenograft Models of Ovarian Cancer for Therapy Evaluation.

The evaluation of novel treatment regimes in ovarian cancer, ranging from cytotoxic agents and targeted therapy to surgery, demands clinically relevant mouse models to mimic human disease. These more advanced preclinical models provide a tool to obtain robust data on the mechanism of action, cytotoxicity and therapeutic efficacy of newly emerging antitumor therapies.In this chapter, we describe how to generate ovariancancer xenograft models through injection of human tumor cell lines in immunocompromised mice. Detailed methodological descriptions are provided for both the commonly applied subcutaneous model and the more technically challenging orthotopic tumor model that involves inoculation of cancer cells in the ovarian bursa. We demonstrate how to monitor tumor growth and metastases in orthotopic ovarian models through noninvasive optical imaging and the procedures for treatment strategy, including administration of test compounds and debulking surgery. We comment on the strengths, limitations, and procedural challenges associated with each of the models.

I.p.-injected cationic liposomes are retained and accumulate in peritoneally disseminated tumors

Intraperitoneal (i.p) chemotherapy is an attractive approach to treat peritoneally disseminated cancers by delivering therapeutic agents directly to the peritoneal cavity where some disseminated tumors are located. Cationic liposomes (CLs) have been used as a viable delivery carrier for i.p. chemotherapy to improve the peritoneal retention of anticancer agents. However, there are no reports on the fate of CLs following i.p. administration to the peritoneal cavity in the presence of disseminated tumors. We prepared a tumor xenograft murine model of peritoneally disseminated gastric cancer by i.p. inoculation of human gastric cancer cells and followed the fate of either CLs or PEGylated CLs (PEG-CLs) after i.p. injection in the model. I.p.-injected CLs were retained in peritoneal cavity for at least 3 days post-injection as a result of clustering with ascites fluid proteins, mainly albumin, while i.p. PEG-CLs was rapidly cleared from the peritoneal cavity to the circulation within 3 h post-injection. Importantly, i.p. CLs efficiently accumulated in the targeted disseminated tumor cells, but not in other abdominal organs including liver, spleen, and kidney. The tumor selectivity upon i.p. administration of CLs may be associated with the lymphatic drainage system. A lipoplex formulation composed of CLs with short hairpin RNA (shRNA) against luciferase, a model therapeutic agent, suppressed luciferase activity in peritoneally disseminated tumors by 80%, with no cytokine secretion in serum. This suggests that i.p. CLs can efficiently deliver a therapeutic agent to peritoneally disseminated tumors with few systemic adverse events. These results suggest that i.p. treatment with CLs or non-PEGylated lipoplexes may be a promising approach for the treatment of peritoneally disseminated cancers through their ability to selectively deliver therapeutic agents to i.p. target sites with minimal systemic adverse events.

Abstract 1806: Novel preclinical orthotopic and bone metastasis TNBC models for assessment of immunotherapy and chemotherapy responses

Abstract Triple-negative breast cancer (TNBC) is one of the most complicated types of breast cancer to treat. However, clinical trials continue to demonstrate that combining immunotherapy with chemotherapy can improve survival in patients with metastatic TNBC. In preclinical immunotherapy development, one of the challenges and limitations is the lack of representative TNBC models for advanced metastatic disease that could be used to study survival benefit of these compounds. The aim of this study was to develop local and bone metastatic TNBC models where survival could be followed as primary endpoint, and to assess effects of standard-of-care (Nab-paclitaxel, Abraxane) and anti-PD-1 (pembrolizumab, Keytruda). Age-matched immunodeficient female CIEA NOG® mice (NOG) and NOG mice engrafted with human CD34+ hematopoietic stem cells (huNOG) were used in the study. In the orthotopic model, the luciferase labelled human TNBC cells, MDA-MB-231(SA)-luc, were inoculated into mammary fat pad of the mice. In the intratibial model, MDA-MB-231(SA)-luc cells were inoculated into bone marrow cavity of tibia to model the growth of primary and bone metastatic TNBC. The treatment with vehicle or paclitaxel (10 mg/kg) in NOG mice or isotype control or pembrolizumab (5 mg/kg) in huNOG mice was started 3 days after cancer cell inoculation and continued twice a week until termination. The mice were sacrificed individually according to predefined sacrifice criteria or latest at study week 8. Nab-paclitaxel prolonged survival of immunodeficient mice with both primary and metastatic TNBC, but the effect was more profound in the orthotopic model. Pembrolizumab treatment in humanized mice with either tumor site did not result in survival benefit. In an earlier study, we have observed modest tumor growth inhibition with pembrolizumab in the orthotopic model suggesting donor variation effect. The findings are consistent with the clinical studies where pembrolizumab as monotherapy has demonstrated benefit for only some of the TNBC patients. In summary, novel preclinical models mimicking primary and bone metastatic TNBC were established that could be used to study the survival benefit of developing new treatments, including immunotherapies, against local and advanced TNBC. Citation Format: Justyna Zdrojewska, Ivan Gladwyn-Ng, Jenni H. Mäki-Jouppila, Katja M. Fagerlund, Mari I. Suominen, Ross Horn, Jukka Rissanen. Novel preclinical orthotopic and bone metastasis TNBC models for assessment of immunotherapy and chemotherapy responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1806.

Novel bone-targeted parathyroid hormone-related peptide antagonists inhibit breast cancer bone metastases

Patients with advanced breast cancer often develop bone metastases. Treatment is limited to palliative care. Parathyroid hormone (PTH)/parathyroid hormone-related peptide (PTHrP) antagonists for bone metastases failed clinically due to short half-life and inadequate concentration in bone. We synthesized two novel PTHrP antagonists fused to an inert bacterial collagen binding domain (CBD) that directs drugs to bone. PTH(7-33)-CBD is an N-terminal truncated PTHrP antagonist. [W2]PTH(1-33)-CBD is an PTHrP inverse-agonist. The aim of this study was to assess PTH(7-33)-CBD to reduce breast cancer bone metastases and prevent osteolytic destruction in mice and to assess both drugs for apoptosis of breast cancer cells in vitro and inhibition of PTH receptor (PTHR1). PTH(7-33)-CBD (1000 µg/kg, subcutaneous) or vehicle was administered 24 h prior to MDA-MB-231 breast cancer cell inoculation into the tibia marrow. Weekly tumor burden and bone density were measured. Pharmacokinetic analysis of PTH(7-33)-CBD in rat serum was evaluated. Drug effect on cAMP accumulation in SaOS-2 osteosarcoma cells and apoptosis of MDA-MB-231 cells was assessed. PTH(7-33)-CBD reduced MDA-MB-231 tumor burden and osteolytic destruction in mice 4-5 weeks post-treatment. PTH(7-33)-CBD (1000 μg/kg i.v. and subcutaneous) in rats was rapidly absorbed with peak concentration 5-min and terminal half-life 3-h. Bioavailability by the subcutaneous route was 43% relative to the i.v. route. PTH(7-33)-CBD was detected only on rat periosteal bone surfaces that stained positive for collagen-1. PTH(7-33)-CBD and [W2]PTH(1-33)-CBD (10-8M) blocked basal and PTH agonist-induced cAMP accumulation in SaOS-2 osteosarcoma cells. Both drugs induced PTHR1-dependent apoptosis of MDA-MB-231 cells in vitro. Novel bone-targeted PTHrP antagonists represent a new paradigm for treatment of breast cancer bone metastases.

Abstract PO072: Nitric oxide tumor ablation stimulates an anti-tumor immune response in mice

Abstract Introduction: Metastases are responsible for a major portion of the morbidity and mortality of cancer, accounting for approximately 90% of all cancer-related deaths. In situ destruction of the tumor mass has been reported to provide the immune system with an antigen source for the induction of antitumor immunity, which can destroy distant metastases. One of the proposed mechanisms involves the induction of antigen presenting cells that may result in the stimulation of adaptive immunity. Our research group is developing an innovative gaseous Nitric Oxide (gNO) based tumor ablation method. NO is a short-lived free radical which, at high doses, possesses anticancer properties. Moreover, NO has been proven to activate innate and adaptive responses of the immune system against tumors. Previous in vivo results showed that all gNO-treated colon tumor-bearing (CT26 model) mice (n=6) were resistant to a secondary CT26 cell inoculation. In the current study, high dose gNO has been used to destroy breast and lung solid tumors in mice. The immune response stimulated following this treatment was tested. Materials and Methods: Breast and lung tumor-bearing mice (4T1 and LLC1 models, respectively) were treated with 50,000 ppm gNO intratumorally. A metastasis model was induced in all gas-treated tumor bearing mice up to 14 days post treatment by challenging the mice with a second cancer cell inoculation (challenge assay). Naïve mice, inoculated with the same cancer cells, served as an internal control. Results: All gNO-treated lung tumor-bearing mice (n=2) were resistant to a second LLC1 lung cancer cell inoculation as compared to 100% tumor take in the control group (n=3, p = 0.03). In gNO-treated breast tumor-bearing mice (n=3), using the highly aggressive 4T1 model that mimics human stage 4 breast cancer, an inhibition in a secondary 4T1 tumor take was observed as compared to the tumor take in the control group (n=3). All naïve mice that were inoculated with 4T1 cells developed a tumor after 5 days, while no gNO-treated 4T1 tumor-bearing mice developed a 4T1 challenge tumor at this time point (p =0.01). At 7 days after 4T1 challenge cell inoculation 1 of 3 mice developed a challenge tumor (p =0.08). Conclusion: The proposed project represents development of a novel treatment for cancer. Our innovative gNO based treatment may serve to treat lethal solid tumors locally and their distant metastases systemically via the stimulation of an anti-tumor immune response. Citation Format: Hila Confino, Shay Yarkoni, Matan Goldshtein, Elya Dekel, Omer Lerner, Shani Puyesky, Steve Lisi, Rinat Kalaora, Pam Golden, Amir Avniel, Prof. Ido Wolf. Nitric oxide tumor ablation stimulates an anti-tumor immune response in mice [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO072.

Abstract 1634: Orthotopic and bone metastasis prostate cancer models using the 22Rv1 cell line

Abstract Prostate cancer is the most common cancer in men. Upon diagnosed malignancy the growth of the tumors is androgen driven in a majority of the cases. For that reason, the tumors are treated with androgen deprivation therapy (ADT) to stop or reduce tumor growth. Later, the tumors often become resistant to ADT and at this stage the disease is described as castration-resistant prostate cancer (CRPC). Bone metastasis is one of the hallmark of disease progression. Metastatic disease can be either androgen sensitive or castration resistant (mCRPC) in which the mortality rate is over 50%. Prostate cancer metastases are commonly formed in bones. The 22Rv1 human prostate carcinoma cell line is androgen sensitive and shown to express androgen receptor (AR). The 22Rv1 cells are an invaluable model system to study AR function, the efficacy of existing drugs and to design novel anti-AR therapies that also target non-truncated regions of AR. The aim of this study was to establish predictive orthotopic and intratibial preclinical in vivo prostate cancer models that can be used in drug development when targeting cancer cells and their local environment. Male athymic nude mice (5-6 weeks old) and NOG mice (over 20 weeks old) were used in the study. 2.5 × 105 22Rv1 cells were inoculated orthotopically into the prostate of NOG mice, and 0.5 × 106 cells were inoculated intratibially into the bone marrow cavity of athymic nude mice. 22Rv1 human prostate cancer cells (ATCC) were cultured in RPMI 1640 medium supplemented with 10% iFBS, 2 mM L-Glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4500 mg/l D-Glucose and P/S. Tumor growth was followed by PSA measurements using Human PSA ELISA assay. In addition, in the intratibial model, tumor-induced bone changes were monitored by X-ray imaging of the hind limbs. Prostates and hind limbs were collected at sacrifice, fixed in 10% NBF, and processed to paraffin blocks for further histological analysis. Sections of prostate were stained with hematoxylin-eosin (HE) and AR. Tumor-bearing and contralateral (healthy) tibias were stained with HE - OrangeG, MGT, TRAP, and AR. For enhanced visualization of tumor models in 3D, we reconstructed the tissue from serial sections using a tailored computational pipeline based on applying a deformable model for corresponding points from adjacent sections. The observed tumor take rate was 100% in both models. Confirmed by IHC, tumors formed in both the orthotopic and intratibial models expressed AR. 22Rv1 cells formed osteoblastic - lytic mixed bone lesions in the intratibial model. Based on lesion areas, randomization of the tumor bearing mice in the intratibial model can be done after 2 weeks from cancer cell inoculation. In conclusion, both presented models are suited to test the anticancer efficacy of new drug candidates. In addition, the intratibial model can be used to test the efficacy of novel drug candidates on prostate cancer cells in bone metastatic microenvironment. Citation Format: Justyna Zdrojewska, Tiina E. Kähkönen, Jenni H. Mäki-Jouppila, Masi Valkonen, Pekka Ruusuvuori, Jussi M. Halleen, Jenni Bernoulli. Orthotopic and bone metastasis prostate cancer models using the 22Rv1 cell line [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1634.

Abstract 3317: Discovery of novel STING agonists with robust anti-tumor activity

Abstract Introduction: Stimulator of interferon genes (STING) is an endoplasmic reticulum (ER) membrane signaling protein, which plays a key role in the sensing of cytoplasmic DNA arising from bacterial and viral infection, as well as self-DNA. Several studies have shown that STING signaling is essential for antitumor immunity by inducing the production of type I IFN and activating both innate and adaptive immunity. Therefore, activation of STING pathway in the tumor microenvironment could be a potential effective approach for cancer immunotherapy. Here, we present the characterization of novel STING agonists in a variety of in vitro cell lines and in vivo murine models. Methods: THP-1 monocyte cells and HEK 293-derived cells transfected with STING variants were used for cellular potency evaluation. Multiple murine tumor models were used for evaluation of in vivo efficacy. Tumor cells were inoculated into C57BL/6 mice and compounds were injected intratumorally three times at every three days when tumors reached a mean volume of approximately 100 mm3. Compounds-treated mice free from tumor for 30 days were re-challenged with MC38 tumor cells in MC38 murine model. Results: Several lead compounds CS-1018, CS-1020 and CS-1010 activate mouse and human STING variants in vitro with much better potency than natural ligand cGAMP and reference compound X. CS-1018 demonstrated dose dependent robust antitumor activity in B16F10 and MC38 mouse models. 3 out of 8 mice achieve complete responses at the dose of 100 ug in B16F10, 25% or 75% of mice were tumor free when treated with 5 ug or 25 ug of compound respectively in MC38. The survival mice from 5 ug and 25 ug groups remained tumor-free after re-challenged with another inoculation of MC38 cancer cells. The antitumor activity of compound CS-1020 and CS-1010 was also demonstrated in MC38 mouse model. When CS-1020 was dosed at 5 ug or 25 ug, complete responses were observed in 63% or 88% of mice respectively. In the case of CS-1010, the dose of 5 ug provides complete responses in 100% of treated mice. Similarly, both compounds induced immune-mediated tumor rejection when re-challenged with the same cancer cells. Conclusions: We have identified several STING agonists, which demonstrated better cellular potency to mouse and human STING alleles compared to natural ligand cGAMP and reference compound. All compounds showed dose dependent antitumor activity in MC38 or B16F10 syngeneic models, in addition, tumor-free mice after treatment of compounds developed tumor specific immunologic memory response in MC38 murine model. Combination studies of identified STING agonists with other IO agents are currently ongoing. The results will be discussed. Citation Format: Anrong Li, Yuntao Song, Chen Dong, Xiaoqi Chen, Junbao Yang. Discovery of novel STING agonists with robust anti-tumor activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3317.

Abstract 2838: Peritoneal immune evasion can be overcome with STING agonist treatment in peritoneal carcinomatosis of colon cancer

Abstract Introduction: Peritoneal immune system plays an important role during the peritoneal dissemination of colon cancers. Therefore, tumor-induced T cell anergy in peritoneal cavity could lead to immune evasion and the progression of peritoneal carcinomatosis. Here, we aimed to reverse the immune evasion in peritoneal cavity and effectively suppress the peritoneal carcinomatosis of colon cancer by STING-based immunotherapy. Methods: We established an animal model of peritoneal carcinomatosis by intraperitoneal injection of MC38 colon cancer cells to C57BL/6 mice. Temporal changes in peritoneal immune microenvironment during peritoneal tumor dissemination were first characterized by analyzing tumor nodules, malignant ascites, and peritoneum lavage by flow cytometry. Then, tumor-bearing mice were treated with either STING agonist (ADU-S100), and/or anti-PD-1 antibody. Tumor burden and the volume of ascites were assessed after the treatment. The tumors were analyzed with flow cytometric, histologic, and RNA sequencing analyses. Results: The intraperitoneal inoculation of MC38 colon cancer cells led to dramatic immune cell changes in peritoneal cavity. While the number of myeloid-derived suppressor cells (MDSCs) were markedly increased, the number of CD8+ T cells and dendritic cells gradually decreased after initial influx in the peritoneal cavity. Treatment with STING agonist remarkably suppressed the growth of peritoneal seeding nodules (75%). Moreover, normalization and reduction of peritoneal tumor blood vessels resulted in an overt decrease in malignant ascites (85%) in the peritoneal cavity after STING agonist treatment. In addition, the number of dendritic cells and CD8+ T cells had markedly increased, but that of MDSCs was decreased in the peritoneal cavity. Moreover, the number of IFN-gamma-secreting cytotoxic T cells were increased in tumor nodules and peritoneal cavity after STING agonist treatment. Finally, the combination immunotherapy of STING agonist and anti-PD1 antibody induced stronger adaptive immunity against peritoneal carcinomatosis than monotherapy and showed potent anti-cancer activity with reduced ascites formation. Conclusion: STING immunotherapy can normalize peritoneal tumor vasculature and immune system, and synergize with immune checkpoint blockade to control peritoneal carcinomatosis of colon cancer. Citation Format: Hannah Yang, Seung Jun Lee, Yu Seong Lee, Won Suk Lee, So Jung Kong, Beodeul Kang, Hong Jae Chon, Chan Kim. Peritoneal immune evasion can be overcome with STING agonist treatment in peritoneal carcinomatosis of colon cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2838.

PD-1 blockade inhibits osteoclast formation and murine bone cancer pain.

Emerging immune therapy, such as with the anti-programmed cell death-1 (anti-PD-1) monoclonal antibody nivolumab, has shown efficacy in tumor suppression. Patients with terminal cancer suffer from cancer pain as a result of bone metastasis and bone destruction, but how PD-1 blockade affects bone cancer pain remains unknown. Here, we report that mice lacking Pdcd1 (Pd1-/-) demonstrated remarkable protection against bone destruction induced by femoral inoculation of Lewis lung cancer cells. Compared with WT mice, Pd1-/- mice exhibited increased baseline pain sensitivity, but the development of bone cancer pain was compromised in Pd1-/- mice. Consistently, these beneficial effects in Pd1-/- mice were recapitulated by repeated i.v. applications of nivolumab in WT mice, even though nivolumab initially increased mechanical and thermal pain. Notably, PD-1 deficiency or nivolumab treatment inhibited osteoclastogenesis without altering tumor burden. PD-L1 and CCL2 are upregulated within the local tumor microenvironment, and PD-L1 promoted RANKL-induced osteoclastogenesis through JNK activation and CCL2 secretion. Bone cancer upregulated CCR2 in primary sensory neurons, and CCR2 antagonism effectively reduced bone cancer pain. Our findings suggest that, despite a transient increase in pain sensitivity following each treatment, anti-PD-1 immunotherapy could produce long-term benefits in preventing bone destruction and alleviating bone cancer pain by suppressing osteoclastogenesis.

1095-P: Exendin 4 Decreases Tumor Volume through Attenuation of psma-Induced Activation of NfκB Pathway in HPV-Induced Diabetes/Cancer Model

Background: The diabetes-cancer link may be mediated by inflammation due to infection such as Human Papilloma Virus (HPV). Exendin 4, a Glucagon-like-peptide 1 agonist, has anti-hyperglycemic and anti- inflammatory effect and can attenuate certain cancer growth. Methods: We used RNA microarray analysis to detect the gene expression after Exendin 4 treatment in the db/db mouse tumor, which was established by inoculation of mouse PV cancer cells (CUP-1). The latter originates from the kidney of baby C57BL/6J mice expressing HPV-16 E7 oncogene. We examined the effect of Exendin 4 on cell proliferation, apoptosis and gene expression along the inflammatory pathway in CUP-1 under different glucose environment. We developed CUP-1 cells with or without target gene overexpression (OE) or downregulation (DR) for inoculation into the db/db mouse. We compared RNA expression of the target gene in cervical cancer specimen from patients with or without diabetes. Results: RNA expression of proteasome 20s (psma) was decreased after Exendin 4 treatment in the db/db mouse tumor. High glucose environment increased cell proliferation in CUP-1 cells, which was enhanced after OE of psma but attenuated after DR of psma. Exendin 4 suppressed NFκB P65 and IκB activation in CUP-1 cells under high glucose medium. After inoculation of CUP-1 cells with DR and OE of psma, the tumor size was decreased and increased in db/db mice respectively. Exendin 4 decreased the tumor volume in the db/db mice of psma OE group. In human cervical cancer specimen, RNA expression of psma was increased in patients with diabetes versus those without diabetes. Conclusion: High glucose increased expression of psma, which activated NFκB inflammation pathway and promoted carcinogenesis. Exendin 4 suppressed the NFκB pathway activation and decreased tumor volume with corroborative evidence from human samples, suggesting that the anti-cancer effect of Exendin 4 might be mediated by the psma-NFκB pathway. Disclosure D. Mao: None. J.C. Chan: None.

CCL8 Promotes Postpartum Breast Cancer by Recruiting M2 Macrophages.

SummaryThe microenvironment of postpartum mammary gland promotes tumor growth and metastasis in animal models and is linked to increased risk of breast cancer and poor disease outcome in patients. Our previous studies showed the involvement of the chemokine CCL8 in breast cancer metastasis through modulation of the tumor-promoting activity of the tumor microenvironment. Here we show that CCL8 is highly expressed during mammary gland involution and enhances the infiltration of M2 subtype macrophages at the second phase of involution. Cancer cell inoculation studies in Ccl8-deficient animals indicate that CCL8 accelerates tumor onset during involution but not in nulliparous animals. Depletion of macrophages abolished the tumor-promoting effect of CCL8 in involution suggesting the specific role of CCL8 in promoting tumor growth by recruiting macrophages. These results underscore the role of CCL8 in the development of postpartum breast cancer and suggest the potential value of targeting CCL8 in disease management.

CD40 signaling induces type I interferon and immune control in mouse pancreatic cancer lacking the CXCL12-coat

Abstract Pancreatic ductal adenocarcinoma (PDA) has a low 5 year survival rate of 8.5% because of its late diagnosis and resistance to the existing therapies. Patients with PDA who are microsatellite stable do not respond to check point blockade immunotherapy due to impaired T cell infiltration, which is mediated by the CXCL12-coat on cancer cells (Wang Z, et al. bioRχiv). In mouse hepatic metastases established with PDA cells from the KPC model of PDA, Krt19-CRISPR/Cas9-edited tumors lacking the CXCL12-coat showed a type I interferon (IFN) response as well as T cell infiltration and activation compared to the control scramble tumors. By RNA FISH, we found that IFNb1 is dominantly produced by CD40+ cells, and not by SiglecH+ plasmacytoid dendritic cells. RNA sequencing data showed up-regulation of Cd40lg in the Krt19-edited tumors compared to the scramble tumors. Administration of antagonistic anti-CD40 antibody prior cancer cells inoculation abolished IFNb1 production, T cell accumulation and activation. To confirm the role of CD40, administration of agonistic anti-CD40 antibody to mice bearing wild type PDA tumors elicited intra-tumoral IFNb1 production in CD11b+ CD11c+ myeloid cells. The T cell infiltration and activation induced by agonistic anti-CD40 antibody treatment sensitized both subcutaneous pancreatic tumors and hepatic metastases to anti-PD1 treatment. These findings indicate that the expression of CD40L elicits an intra-tumoral type I IFN response that is required for immune control of PDA tumors in which CXCR4 signaling is impaired.

Early intervention of bone-nearby acupuncture combined with electroacupuncture on morphine tolerance in bone cancer pain rats and its effect on the expression of HDAC and MOR in dorsal root ganglia

To observe the effect of early intervention of bone-nearby acupuncture (BNA) combined with electroacupuncture (EA) on the expression of histone deacetylase1(HDAC1), histone deacetylase 2 (HDAC2) andμ-opioid recepter (MOR) in dorsal root ganglia (DRG) of bone cancer pain-morphine tolerance (BCP-MT) rats, and to explore its possible mechanism. A total of 35 SD rats were randomized into a sham BCP group (n=6), a BCP group (n=7), a MT group (n=7), a BNA+EA group (n=8) and a shame BNA group (n=7). Except of the sham BCP group, cancer cell inoculation operation at left tibia was given in the other 4 groups to establish the bone cancer pain model. In the MT group, the BNA+EA group and the shame BNA group, intraperitoneal injection of morphine hydrochloride was given to establish the morphine tolerance model. After the operation, bone-nearby acupuncture combined with electroacupuncture was applied at "Zusanli" (ST 36) and "Kunlun" (BL 60) in the BNA+EA group, with dilatational wave, 2 Hz/100 Hz in frequency, 0.5 to 1.5 mA in intensity. Intervention in the shame BNA group was applied at the same time and acupoints as those in the BNA+EA group, the needles were pierced the skin without any electrical stimulation. The needles were retained for 30 min, once a day for continuous 7 days in both BNA+EA and shame BNA groups. Before and 10, 11, 15, 22 days after the operation, the left paw withdrawal threshold (PWT) was measured in the 5 groups. The levels of HDAC1, HDAC2 and MOR in DRG were detected by Western blot. Ten days after the cancer cell inoculation operation, the PWT of the BCP, MT, BNA+EA and sham BNA groups was decreased compared with the sham BCP group (P<0.01). Eleven days after the operation, the PWT of the MT, BNA+EA and sham BNA groups was increased compared with the BCP group (P<0.01). Twenty-two days after the operation, the difference was no significant between the BCP group and MT group (P>0.05); the PWT of the BNA+EA group was increased compared with the MT and sham BNA group (P<0.01). In the BCP group, the DRG levels of HDAC1 and HDCA2 were increased, while the level of MOR was decreased compared with the sham BCP group (P<0.05, P<0.01). In the MT group, the DRG level of HDAC1 was increased compared with the BCP group (P<0.05). In the BNA+EA group, the DRG level of HDAC1 was decreased compared with the MT group and the sham BNA group (P<0.01, P<0.05), while the level of MOR was increased (P<0.01). Early intervention of bone-nearby acupuncture combined with electroacupuncture can relieve the morphine tolerance in bone cancer pain rats, it may relate to down-regulating the expression of HDAC1 and up-regulating the expression of MOR in the dorsal root ganglia.

Unexpected effects of long-term treatment with acetylsalicylic acid on late phase of pulmonary metastasis in murine model of orthotopic breast cancer.

Long-term administration of acetylsalicylic acid (ASA) was effective in prevention of colorectal cancer, whereas the efficacy of this compound in other cancer types, including breast cancer, has been less convincingly documented. Indeed, the antimetastatic effect of low-dose ASA was observed only in the early intravascular phase of metastasis of breast cancer. In the present work, we characterized the effects of long-term treatment with ASA on the late phase of pulmonary metastasis in a mouse orthotopic 4T1 breast cancer model. Mice were treated with ASA at a dose of 12 mg·kg-1 of body weight daily starting one week prior to inoculation of 4T1 breast cancer cells, and the treatment was continued throughout progression of the disease. ASA administration decreased platelet TXB2 production in ex vivo assays but did not change thrombin-induced platelet reactivity. Although the number of metastases in the lungs remained unchanged in ASA-treated mice, infiltration of inflammatory cells was increased concomitantly with higher G-CSF and serotonin concentrations in the lungs. Pulmonary NO production was compromised compared to control 4T1 mice. ASA treatment also evoked an increase in platelet and granulocyte counts and decreased systemic NO bioavailability along with increased markers of systemic oxidant stress such as higher GSSG/lower GSH concentrations in RBC. Analysis of eicosanoids in stirred blood demonstrated that administration of ASA at a dose of 12 mg·kg-1 to cancer-bearing mice had an effect beyond inhibition of platelet COX-1, suggesting long-term treatment with low-dose aspirin is not a selective murine platelet COX-1/TXA2 pathway inhibitor in cancer-bearing mice. In summary, quite surprisingly, long-term treatment with low-dose ASA administered until the advanced phase of breast cancer in a murine orthotopic model of 4T1 breast cancer negatively affected the phenotype of the disease.

Bone-edge electroacupuncture relieves pain by regulating expression of GRK5, β-arrestin 2 and PKCα proteins in locus coeruleus in bone cancer pain rats with morphine tolerance

To observe the effect of bone-edge electroacupuncture (EA) intervention on mechanical pain threshold (PT) and expression of G protein-coupled receptor kinase (GRK5), β-arrestin 2, total and phosphorylated PKC alpha (p-PKCα) proteins in the locus coeruleus (LC) of rats with bone cancer pain induced morphine tolerance, so as to reveal its partial central mechanisms underlying pain relief. Forty SD rats were randomly divided into 5 groups, namely sham bone cancer, bone cancer pain, morphine tolerance, bone-edge EA, and sham EA (n＝ 8 rats in each group). The bone cancer with morphine tolerance model was established by intramedullary injection of MRMT-1 cells into the tibial cavity, and then intraperitoneal injection of morphine hydrochloride injection. After successful establishment of morphine tolerance model, the bone-edge EA (2 Hz/100 Hz，0.5－1.5 mA) was applied to bilateral "Zusanli" (ST36) and "Kunlun" (BL60) for 30 min, once a day for 7 days, after inserting the needle-tip to the tibial bone surface. The ipsilateral mechanical paw withdrawal thresholds (PWTs) were detected dynamically. The expression levels of GRK5, β-arrestin 2, PKCα and p-PKCα in the LC area were measured by Western blot. The PWTs of bone cancer pain rats were decreased on day 10 after inoculation of cancer cells (P<0.01). After i．p. of morphine for 11 days, no analgesic effect and pain tolerance appeared (P>0.05). The PWTs were significantly increased in the bone-edge EA intervention group (P<0.01), not in the sham EA group (P>0.05). In comparison with the sham bone cancer group, the expression of GRK5 protein in morphine tolerance group was significantly decreased (P<0.01); compared with morphine tolerance group, the expression of GRK5 protein in bone-edge EA group was increased(P<0.01). In comparison with the sham bone cancer group, the expression of β-arrestin 2 and p-PKCα in bone cancer group significantly increased (P<0.01). After the intervention, the increased β-arrestin 2 and p-PKCα expressions were reversed in the bone-edge EA group (P<0.01); compared with morphine tolerance group and sham EA group, the expression of PKCα protein was decreased(P<0.01). Bone-edge EA can effectively relieve morphine tolerance in bone cancer pain rats, which may be related to its functions in up-regulating GRK5 protein and down-regulating β-arrestin 2, PKCα and p-PKCα proteins in LC. .

Podoplanin promotes cancer-associated thrombosis and contributes to the unfavorable overall survival in an ectopic xenograft mouse model of oral cancer

BackgroundPodoplanin (PDPN) is a transmembrane glycoprotein that mediates tumor cell-induced platelets aggregation in different cancer types. Emerging data indicate that PDPN is a marker for poor prognosis of human oral squamous cell carcinoma (OSCC). However, the functional impacts of PDPN on cancer formation and disease progression of OSCC remain to be elucidated. MethodsThe sublines of the OECM-1 oral cancer cells with PDPN knockdown or overexpression were established. The cellular characteristics and the ability to induce platelet aggregation of these cells lines were analyzed. An ectopic xenograft animal model by inoculating cancer cells into the anterior neck region of nude mice was established to investigate the functional impact of PDPN on disease progression and cancer-associated thrombosis of OSCC. ResultsPDPN promoted OSCC cell migration and invasion, but had no effect on cell proliferation in vitro and tumor growth in vivo. Co-incubation of PDPN-positive (PDPN+) OSCC cells with platelets induced platelet activation and aggregation. The mice bearing PDPN+ tumor had a decrease in overall survival despite that there was no gross appearance of distant metastasis. A speckled immunofluorescence staining pattern of platelet marker mCD41 was defined in the PDPN+ tumor sections and the intensity was greater than in the PDPN-low or negative tumor sections. Co-immunofluorescence staining of the tumor sections with mCD41 and the endothelial cell marker mCD31 further demonstrated that platelet aggregates were located in the lumen of blood vessel and were also distributed intratumorally in the mice bearing PDPN+ tumors. ConclusionsThese data demonstrated that PDPN expression in the cancer cells is associated with high risk of thrombosis, leading to unfavorable overall survival of the mice. This study provides new insights into the functions of PDPN in cancer-associated thrombosis and in the pathophysiology of OSCC.

Abstract B143: Establishment of primary tumor models of TNBC and ER+/HER2- breast cancer in humanized mice and validation of pembrolizumab efficacy

Abstract Background: Immunotherapy is an emerging treatment option for different subtypes of breast cancer. Pembrolizumab (Keytruda®) targets PD-1 expressing T cells in tumors and mediates anti-tumor effects. Targeting PD-1 has shown promising results in advanced breast cancer and is being evaluated in clinical trials in triple-negative breast cancer (TNBC) and in estrogen receptor positive, human epidermal growth factor 2 negative (ER+/HER2-) breast cancer patients. Immunotherapy is not yet an established treatment option for early breast cancer. The aim of this study was to establish preclinical primary tumor models for TNBC and ER+/HER2- in humanized mice, and to evaluate efficacy of pembrolizumab in the models. Materials and methods: Immunodeficient CIEA NOG female mice engrafted with human CD34+ hematopoietic stem cells (huNOG mice) were inoculated orthotopically into mammary fat pad with either MDA-MB-231(SA) (TNBC) or MCF-7 (ER+/HER2-) human breast cancer cells. The mice receiving MCF-7 cells were implanted with estradiol (E2) releasing pellets before cancer cell inoculation to support tumor growth. The mice were treated with pembrolizumab (5 mg/kg) or isotype control in a Q5D schedule. The treatments were started 3 or 14 days after cancer cell inoculation in the TNBC and ER+/HER2- models, respectively. Tumor growth was monitored by caliper measurements, and the study was terminated at 3 weeks (TNBC model) or 7 weeks (ER+/HER2- model). Tumors were collected and analyzed immunohistochemically for tumor-infiltrating lymphocytes (TILs), and hematological analysis was performed from the mice in the ER+/HER2- model. Results: Tumors responded to pembrolizumab treatment in the TNBC model. Three out of eight mice responded to the treatment and one of them had a tumor rejection. Pembrolizumab treatment was associated with decrease in CD8+ TILs and granzyme B+ cells in the tumor. In the ER+/HER2- model, E2 supplementation was essential for tumor growth. However, E2 caused a decrease in the clinical condition, including decrease in white and red blood cells, leading to early sacrifices. Only modest responses for pembrolizumab were observed in a few mice and the results did not reach statistical significance. No major changes were observed in TILs due to pembrolizumab treatment. Conclusions: In the established breast cancer models in humanized mice, pembrolizumab decreased tumor growth in the TNBC model but induced only minor tumor responses in the ER+/HER2- model. Estrogen was essential for ER+/HER2- tumor growth but induced severe anemia and had immunomodulatory effects potentially influencing the lack of pembrolizumab efficacy in the model. Citation Format: Tiina E Kähkönen, Mari I Suominen, Jenni HE Mäki-Jouppila, Azusa Tanaka, Philip Dube, Michael Seiler, Jussi M Halleen, Jenni Bernoulli. Establishment of primary tumor models of TNBC and ER+/HER2- breast cancer in humanized mice and validation of pembrolizumab efficacy [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B143. doi:10.1158/1535-7163.TARG-19-B143

Expression of E-cadherin and specific CXCR3 isoforms impact each other in prostate cancer

BackgroundCarcinoma cells shift between epithelial and mesenchymal phenotypes during cancer progression, as defined by surface presentation of the cell-cell cohesion molecule E-cadherin, affecting dissemination, progression and therapy responsiveness. Concomitant with the loss of E-cadherin during the mesenchymal transition, the predominant receptor isoform for ELR-negative CXC ligands shifts from CXCR3-B to CXCR3-A which turns this classical G-protein coupled receptor from an inhibitor to an activator of cell migration, thus promoting tumor cell invasiveness. We proposed that CXCR3 was not just a coordinately changed receptor but actually a regulator of the cell phenotype.MethodsImmunoblotting, immunofluorescence, quantitative real-time PCR and flow cytometry assays investigated the expression of E-cadherin and CXCR3 isoforms. Intrasplenic inoculation of human prostate cancer (PCa) cells with spontaneous metastasis to the liver analyzed E-cadherin and CXCR3-B expression during cancer progression in vivo.ResultsWe found reciprocal regulation of E-cadherin and CXCR3 isoforms. E-cadherin surface expression promoted CXCR3-B presentation on the cell membrane, and to a lesser extent increased its mRNA and total protein levels. In turn, forced expression of CXCR3-A reduced E-cadherin expression level, whereas CXCR3-B increased E-cadherin in PCa. Meanwhile, a positive correlation of E-cadherin and CXCR3-B expression was found both in experimental PCa liver micro-metastases and patients’ tissue.ConclusionsCXCR3-B and E-cadherin positively correlated in vitro and in vivo in PCa cells and liver metastases, whereas CXCR3-A negatively regulated E-cadherin expression. These results suggest that CXCR3 isoforms may play important roles in cancer progression and dissemination via diametrically regulating tumor’s phenotype.