Treatment Of Tumor-bearing Mice Research Articles

A Novel Salicylanilide Derivative Induces Autophagy Cell Death in Castration-Resistant Prostate Cancer via ER Stress-Activated PERK Signaling Pathway.

Metastatic castration-resistant prostate cancer (CRPC) is currently incurable. Cancer growth and progression is intimately affected by its interaction with host microenvironment. Cotargeting of the stroma and prostate cancer is therefore an emerging therapeutic strategy for metastatic CRPC. Cancer-induced osteoclastogenesis is known to contribute to CRPC bone metastasis. This study is to extend pharmacologic value of our synthesized LCC03, a derivative of 5-(2',4'-difluorophenyl)-salicylanilide that has previously testified for its osteoclastogenesis activity, by exploring its additional cytotoxic properties and underlying mechanism in CRPC cells. LCC03 was chemically synthesized and examined for cell growth inhibition in a serial of CRPC cell lines. We demonstrated that LCC03 dose-dependently suppressed proliferation and retarded cell-cycle progression in CRPC cells. The classical autophagy features, including autophagosome formation and LC3-II conversion, were dramatically shown in LCC03-treated CRPC cells, and it was associated with the suppressed AKT/mTOR signaling pathways, a major negative regulator of autophagy. Moreover, an expanded morphology of the endoplasmic reticulum (ER), increased expression of the ER stress markers GRP78 and PERK, and eIF2α phosphorylation were observed. Blockage of autophagy and PERK pathways using small molecule inhibitors or shRNA knockdown reversed LCC03-induced autophagy and cell death, thus indicating that the PERK-eIF2α pathway contributed to the LCC03-induced autophagy. Furthermore, treatment of tumor-bearing mice with intraperitoneal administered LCC03 suppressed the growth of CRPC xenografts in mouse bone without systemic toxicity. The dual action of 5-(2',4'-difluorophenyl)-salicylanilide on targeting both the osteoclasts and the tumor cells strongly indicates that LCC03 is a promising anticancer candidate for preventing and treating metastatic CRPC.

Comparing the possible anti-tumor effects of synthetic chalcones and metal complexes in a tumor mouse model

Introduction: Traditional treatment of cancer by using chemotherapy not only kills cancer cells but also, normal cells and failed in preventing the spread of the tumor. Chalcone have several biological activities, such as antioxidant, antibacterial, antifungal, anti-inflammatory, and anti-tumor. Aim: To compare the antitumor effects of chalcones in Ehrlich ascetic carcinoma (EAC) bearing mice. Materials and Method: Female Swiss albino mice were randomly divided into 6 groups as the following; Group 1 were served as a negative control, group 2 were inoculated with 2.5×105 (EAC) cells, group 3 were inoculated with 2.5×105 EAC cells and then treated with cisplatin (2 mg/kg), group 4 were inoculated with 2.5×105 EAC cells and then treated with chalcone 1 (0.3 ml gm/L), group 5 were inoculated with 2.5×105 EAC cells and then treated with chalcone II (0.3 ml gm/L). After 7 days, all groups of mice were sacrificed to measure the number and cell cycle of tumor cells as well as the number and activation of CD8+ T cells. Results: Treatment of tumor-bearing mice with chalcone I, but not chalcone II induced decreases in the total number of live cells when compared to control tumor-bearing mice, coinciding with significant increases in G0, G1, S and G2 phases of EAC cells which were comparable to the effects of cisplatin. As compared to control tumor-bearing mice, tumor cells harvested from mice treated with chalcone I showed significant increases in the numbers of activated CD8+ T cells. Conclusion: Chalcone I possesses anti-tumor effects by inducing tumor cells arrest and activation of T cells.

Anti-proliferative effect of ZnO NPs against the growth of Ehrlich Solid carcinoma

Background: Zinc oxide nanoparticle (ZnO NPs) has been widely used in biomedical applications and cancer therapy and has been reported to induce a selective cytotoxic effect on cancer cell proliferation. Methods: Female Swiss Albino mice were inoculated subcutaneously with Ehrlich ascites carcinoma (EAC) cells. ZnO NPs or MTX were injected intraperitoneally. Tumor growth inhibition rate (TIR %), increase in life span (ILS%), zinc concentration, lipid peroxidation marker (MDA), glutathione (GSH) contents, and the activity of the antioxidant scavenger enzymes SOD and catalase CAT was examined. Also, DNA fragmentation and histopathological studies of all groups were carried out. Results: Treatment of tumor-bearing mice with ZnO NPs significantly increased (MST), (TIR %), (ILS %) and reduced tumor weight, compared to tumor-bearing controls. Additionally, Zn concentration and DNA fragmentation were significantly increased in tumor tissues. ZnO nanoparticles were also found to induce oxidative stress, evidenced by the generation of reactive oxygen species and depletion of the antioxidants in tumor tissues, with no changes in the liver. Our data demonstrate that ZnO NPs exert distinct effects via the killing of cancer cells. Conclusion: The study findings demonstrated that the ZnO NPs can induce selective cytotoxicity through reactive oxygen spe­cies generation and oxidative stress.

Abstract I03: Immune contexture of human pancreatic cancer

Abstract The concept that leukocytes are critical components of solid tumors is now generally accepted; however, their role(s) in regulating aspects of neoplastic progression, and/or response to cytotoxic, targeted, and/or immune therapy is only beginning to be understood. Utilizing de novo mouse models of organ-specific solid tumor development (mammary, cutaneous, and pancreas carcinomas and mesothelioma), we now appreciate that adaptive leukocytes differentially regulate myeloid cell recruitment, activation, and effector function, and in turn, activated tumor-infiltrating myeloid cells engage tissue-based programs to foster malignancy and repress antitumor immunity by a diversity of mechanisms. Treatment of tumor-bearing mice with therapeutic agents that disrupt lymphocyte-myeloid cell interaction, myeloid cell activation, or myeloid cell functionality generally slow primary tumor growth kinetics when combined with cytotoxic therapy; however, their impact on metastases is variable. Similar to organ-specific regulatory programs co-opted to foster primary tumor growth, regulation of metastatic seeding and outgrowth is also regulated by tissue- and organ-specific mechanisms. Based on this, it stands to reason that therapeutic strategies may not be efficacious in both primary and metastatic locations. To be presented will be our recent insights into organ- and tissue-specific regulation of primary and metastatic cancer development by adaptive and innate immune cells, how systemic regulation of humoral immunity and complement-mediated pathways regulate pro- versus antitumor immune responses, and new studies evaluating how attenuating protumor properties of select lymphoid and myeloid cells can be exploited to enhance therapeutic responses to cytotoxic and immune-based therapy. (LMC acknowledges generous support from the NIH/NCI, the Department of Defense Breast Cancer Research Program, Susan G. Komen Foundation, the Breast Cancer Research Foundation, and the Brenden-Colson Center for Pancreatic Health.) Citation Format: Lisa M. Coussens. Immune contexture of human pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr I03.

Withaferin A ameliorates ovarian cancer-induced cachexia and proinflammatory signaling

BackgroundOvarian cancer is the fifth leading cause of cancer-related deaths amongst women in the United States. Cachexia is the primary cause of death in approximately 30% of cancer patients, and is often evidenced in ovarian cancer patients. We tested the steroidal lactone Withaferin A to examine if it could ameliorate ovarian cancer-induced cachexia.MethodsSix-week-old severely immunodeficient female mice were xenografted with the ovarian cancer cell line A2780 followed by treatment with Withaferin A or vehicle. Changes in functional grip strength were assessed on a weekly basis. Postmortem, H&E staining was performed on skeletal muscle sections and immunofluorescent immunohistochemistry was performed on skeletal muscle and tumor sections. The levels of NF-κB-related proinflammatory cytokines were assessed in the xenografted tumors and in resident host skeletal muscle.ResultsXenografting of the A2780 cell line resulted in a significant rate of mortality, which was attenuated by a therapeutic dosage of Withaferin A. Mice that received vehicle treatment following xenografting exhibited functional muscle decline over the course of the study. The therapeutic dosage Withaferin A treatment attenuated this reduction in grip strength, whereas the supratherapeutic dosage of Withaferin A was found to be toxic/lethal and demonstrated a further decline in functional muscle strength and an increased rate of mortality on par with vehicle treatment. At a histological level, the vehicle treated tumor-bearing mice exhibited a profound reduction in myofibrillar cross-sectional area compared to the vehicle treated tumor-free control group. The atrophic changes induced by the xenografted tumor were significantly ameliorated by treatment with Withaferin A. The combination of functional muscle weakening and induction of myofibrillar atrophy corroborate a cachectic phenotype, which was functionally rescued by Withaferin A. Further, treatment completely abolished the slow-to-fast myofiber type conversion observed in the settings of cancer-induced cachexia. In both host resident skeletal muscle and the xenografted tumors, we report an increase in NF-κB-related proinflammatory cytokines that was reversed by Withaferin A treatment. Finally, we demonstrated that Withaferin A significantly downregulates cytosolic and nuclear levels of phospho-p65, the active canonical NF-κB transcription factor, in xenografted tumors.ConclusionsCumulatively, our results demonstrate a previously overlooked role of Withaferin A in a xenograft model of ovarian cancer. We propose mechanisms by which Withaferin A reduces NF-κB-dependent pro-inflammatory cytokine production leading to an attenuation of the cachectic phenotype in an i.p. xenograft model of ovarian cancer.

Abstract 5021: LNAplusTM antisense oligonucleotides targeting CD39 and CD73 have potent anti-tumor activity by modulating immunosuppressive tumor microenvironment

Abstract The ectonucleotidases CD39 and CD73 have emerged as promising therapeutic targets to enhance antitumor immune responses, as they act in concert to convert extracellular immune-stimulating ATP to immunosuppressive adenosine. CD39 and CD73 are expressed on different immune cells as well as on cancer cells and support the tumor in escaping immune recognition and destruction. Accordingly, in order to enhance immunity against tumors it would be favorable to increase extracellular ATP- and to reduce adenosine concentrations in the tumor microenvironment. As an alternative approach to small molecule inhibitors or antibodies, we developed antisense oligonucleotides (ASOs) using the OligofyerTM tool to suppress the expression of CD39 and CD73. ASOs were synthesized as Gapmers with flanking locked nucleic acids (LNA) to increase stability and affinity to the target RNA. Knockdown efficacy of ASOs on mRNA and protein level was investigated in cancer cell lines and primary human T cells. CD39 and CD73 activity was evaluated by measuring levels of ATP, AMP and adenosine in cell supernatants. As functional readout we analyzed T cell proliferation and viability in presence of extracellular ATP. The impact of ASO-mediated target knockdown in vivo on anti-tumor immune responses was analyzed in a syngeneic mouse tumor model. Unformulated CD39- and CD73-specific ASOs achieved potent target knockdown on mRNA and protein level in tumor cell lines and primary human T cells in vitro. Furthermore, degradation of extracellular ATP or AMP was significantly blocked by CD39- or CD73-specific ASOs while formation of adenosine was suppressed. Supplementation of cell culture medium with ATP impaired proliferation and viability if T cells expressed CD39 or CD73. Notably, ASO-mediated knockdown of CD39 or CD73 reversed this inhibitory effect of ATP. Systemic treatment of mice with CD39- and CD73-specific ASOs resulted in a knockdown of CD39 and CD73 mRNA expression in spleen and liver. Treatment of tumor-bearing mice with CD39-specific ASO led to dose dependent reduction of CD39 protein expression e.g. in Tregs, tumor-associated macrophages and myeloid-derived suppressor cells. Strikingly, the frequency of intratumoral Tregs was considerably diminished in CD39 ASO-treated animals. Moreover, tumor growth was strongly reduced by CD39-specific ASO. Anti-tumor efficacy was improved when combining CD39 ASO with anti-PD-1 treatment. Taken together, targeting of CD39 and CD73 by ASOs represents a very promising state-of-the art therapeutic approach to improve immunity against tumors. Citation Format: Julia Festag, Tamara Thelemann, Abhishek S. Kashyap, Richard Klar, Sandra Kallert, Melanie Buchi, Lisa Hinterwimmer, Monika Schell, Stefanie Raith, Sven Michel, Alfred Zippelius, Frank Jaschinski. LNAplusTM antisense oligonucleotides targeting CD39 and CD73 have potent anti-tumor activity by modulating immunosuppressive tumor microenvironment [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 5021.

Abstract 937: Nr4a transcription factors limit CAR T-cell function in solid tumors

Abstract In cancer immunotherapy, CD19-targeted CAR T cells have exhibited impressive clinical efficacy against B cell leukemias and lymphomas; however, they have been less effective against solid tumors. This is in part because CAR T cells enter a hyporesponsive (“exhausted” or “dysfunctional”) state that is triggered by chronic antigen stimulation and characterized by upregulation of several inhibitory surface receptors and loss of effector function. To identify transcriptional regulators and other candidates contributing to the diminished CAR T cell function in solid tumors, we developed a CAR T cell model in which recipient mice bearing murine melanoma tumors expressing human CD19 were adoptively transferred with CD19-targeted CAR T cells. We demonstrate that in both CD8+ CAR T tumor-infiltrating lymphocytes (TILs) and endogenous CD8+ TILs expressing high levels of PD-1 and TIM3, the Nr4a proteins Nr4a1 (Nur77), Nr4a2 (Nurr1), and Nr4a3 (Nor1) are prominent effectors of the transcriptional program downstream of NFAT: they promote the expression of inhibitory receptors and genes associated with early hyporesponsive state, and limit effector function. Most importantly, treatment of tumor-bearing mice with CD8+ CAR T cells lacking all three Nr4a factors (Nr4aTKO) resulted in tumor regression and prolonged survival. Nr4aTKO CD8+ CAR T TILs displayed a transcriptomic profile characteristic of effector function, including upregulation of granzymes and cytokines, and many of these gene expression changes were associated with altered regulatory element accessibility near effector genes. Our data identify Nr4a transcription factors as major players in the cell-intrinsic program of T cell hyporesponsiveness and point to Nr4a inhibition as a promising strategy for cancer immunotherapy. Citation Format: Joyce Chen, Isaac F. Lopez-Moyado, Hyungseok Seo, Chan-Wang J. Lio, Laura J. Hempleman, Takashi Sekiya, Akihiko Yoshimura, James P. Scott-Browne, Anjana Rao. Nr4a transcription factors limit CAR T-cell function in solid tumors [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 937.

Abstract 3085: A novel salicylanilide derivative inhibits castration-resistant prostate cancer via endoplasmic reticulum stress-triggered PERK autophagic signaling pathway

Abstract Background and Purpose: Metastatic castration-resistant prostate cancer (CRPC) is currently incurable. This study is to extend pharmacological value of our synthesized salicylanilide derivative, that has previously testified for its osteoclastogenesis activity, by exploring its additional cytotoxic properties and underlying mechanism in CRPC cells. Experimental Approach: LCC03, a 5-(2′,4′-Difluorophenyl)-salicylanilide derivatives, was chemically synthesized and examined for cell growth inhibition in a serial of CRPC cell lines. Possible mechanisms involved in the anticancer activity was elucidated based on the changes in pathway-related protein expression and cellular events, together with antagonists for functional validation. The therapeutic efficacy and safety of LCC03 for the treatment of bone metastatic CRPC was evaluated in an experimental animal model. Key Results: LCC03 dose-dependently suppressed proliferation and retarded cell cycle progression in CRPC cells. The classical autophagy features including autophagsomes formation and LC3-II conversation were dramatically shown in LCC03-treated CRPC cells, and it was associated with the suppressed AKT/mTOR signaling pathways, a major negative regulator of autophagy, Moreover, an expanded morphology of the ER, increased expression of the ER stress markers GRP78 and PERK, and eIF2α phosphorylation were observed. Blockage of autophagy and PERK pathways using small molecule inhibitors or shRNA knockdown reversed LCC03-induced autophagy and cell death, thus indicating that the PERK-eIF2α pathway contributed to the autophagy induced by LCC03. Furthermore, treatment of tumor-bearing mice with intraperitoneal administered LCC03 suppressed the growth of CRPC xenografts in mouse bone without systemic toxicity. Conclusion and Implications: LCC03 induces cellular responses toward ER stress-induced autophagy via the PERK-eIF2α pathway that impairs proliferation and cell cycle progression and exhibits cytotoxicity against CRPC cells. The dual action of 5-(2′,4′-difluorophenyl)-salicylanilide on both the osteoclasts and the tumor cells strongly indicates that LCC03 is a promising anticancer candidate for preventing and treating metastatic CRPC. Citation Format: Chia-Ling HSIEH, Hsu-Shan Hung, Kuan-Chou Chen, Teigi Saka, Chih-Ying Chiang, Shian-Ying Sung. A novel salicylanilide derivative inhibits castration-resistant prostate cancer via endoplasmic reticulum stress-triggered PERK autophagic signaling pathway [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 3085.

Abstract 2762: Development of a novel peptide-formed nanoparticle for pancreatic cancer treatment

Abstract Background and objective: Pancreatic ductal adenocarcinoma (PDAC) is the most common malignant tumor of pancreas with an overall 5-year survival rate of less than 5%. Surgical operation is an curable treatment, however, only 10-20% of patients with PDAC are respectable at diagnosis. Taking advantage of the progression in nanoparticle study, we developed a novel peptide-formed nanoparticle (PF-Nano) and investigate its therapeutic effect in PDAC treatment. Methods: Phage display peptide library was used to screen a high-affinity tumor-homing peptide (THP) for pancreatic cancer (PC) cells. Ligation of THP with cell penetrating peptide was used to prepare a tandem peptide, which automatically formed a nanoparticle. We used this PF-Nano as a vehicle to load and deliver siRNA.Dynamic light scattering (DLS) was employed to define nanoparticle size; and fluorescence imaging was applied to assess PF-Nano siRNA delivery efficiency. In vitro, we used MTT assay and colony formation to evaluate cell growth; wound healing assay to detect cell migration, and flow cytometry to evaluate cell cycle and apoptosis. Therapeutic effect was evaluated by measuring final tumor weight in the mice receiving different treatments. qPCR and Western Blot (WB) were used to detect gene expression in mRNA and protein level. Immunoprecipitation in combination with mass spectrometry and immunofluorescence were used to identify THP’s ligands. Results: The screened peptide has a high-affinity with PC cells. DLS identified evenly distributed PF-Nano with a diameter of approximately 230nm. Detection of fluorescence signal from labeled siRNAs in PC cells and tumor tissue suggested that PF-Nano owned the capacity to successfully deliver siRNA in vitro and in vivo. In vitro treatment with PF-Nano significantly suppressed PC cell proliferation, colony formation, and cell migration. Also it caused cycle arrest in S phase (DNA synthesis) and increase in cell apoptosis. Treatment of tumor-bearing mice with PF-Nano by intraperitoneal injection markedly suppressed tumor growth, evidenced by the reduction of tumor weight to 25% relevant to that in control mice without treatment. Immunoprecipitation and mass spectrometry revealed that protein phosphatase 1 catalytic subunit gamma (PP1C) was a potential binding target of the specific THP. Mechanistically, PF-Nano treatment strongly suppressed RAC-alpha serine/threonine-protein kinase (AKT) phosphorylation, suggesting PP1-AKT signal pathway may be responsible for peptide effect in pancreatic tumor. Conclusion: The established PF-Nano is able to specifically deliver siRNAs to PC tumors and therapeutically suppresses PC tumor growth. Citation Format: Qiongling Wang, Kevin F. Staveley-O’Carroll, Guangfu Li. Development of a novel peptide-formed nanoparticle for pancreatic cancer treatment [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 2762.

Abstract 4080: Expansion of CX3CR1+ cytotoxic CD8+ T cells provides a rationale for IL-15 in combination cancer therapy with PD-1 antibody

Abstract Introduction: IL-15 has been used in clinical trials with PD-1 antibody to overcome cancer resistance to PD-1 monotherapy in cancer patients. However, it is not clear what T cell population would be expanded to mediate tumor control in IL-15 in combination with PD-1 antibody. We recently found CX3CR1+ cytotoxic CD8+ T cells, which increased in responders compared with non-responders after anti-PD-1 in melanoma patients, demonstrated an increased transcription of CD122 (IL2RB). Since CD122 is a subunit of IL-15 receptor, we hypothesized that IL-15 in combination with anti-PD-1 therapy may have the ability to expand CX3CR1+ cytotoxic CD8+ T cells for tumor control. Methods and findings: To examine whether IL-15 plus anti-PD-1 expand CX3CR1+ cytotoxic CD8+ T cells in tumor tissues, we treated tumor-bearing mice with IL-15/IL-15Rα complexes alone or with PD-1 antibody and measured the frequency of CX3CR1+Granzyme B+ CD8+ T cells. As only the combination therapy induced dramatic tumor regression, we found the highest increase of CX3CR1+Granzyme B+ CD8 T cells in tumor tissues in the group treated with IL-15 plus anti-PD-1 (28.87±3.19% vs. 7.91±1.32%, p<0.01) compared with groups treated with either IL-15 alone or anti-PD-1 alone. In contrast to wild type mice, IL-15 plus anti-PD-1 did not suppress tumor growth in CX3CR1-KO mice. To determine whether IL-15 alone can also expand human CX3CR1+ cytotoxic CD8+ T cells, we cultured peripheral blood mononuclear cells (PBMCs) isolated from healthy donors (n = 5) with IL-15 for 24 hours in vitro. We found IL-15 (5 ng/mL) only modestly increased the frequency of CX3CR1+ Granzyme B+ CD8+ T cells than those cultured with no IL-15 (39.9±6.44% vs. 27.56±4.98%). To test whether this increase is due to enhanced proliferation of this subset of CD8+ T cells, we measured the expression of Ki-67, a cell proliferative marker, in CX3CR1+ CD8+ T cells by intranuclear staining. We found IL-15 modestly induced more proliferating CX3CR1+ CD8+ T cells in vitro culture compared with control group without IL-15 (6.25±0.54% vs. 0.91±0.25%). Summary: Taken together, we found IL-15 combined with anti-PD-1 therapy has the ability to significantly expand CX3CR1+ cytotoxic CD8+ T cells, however IL-15 alone may have limited ability to significantly expand this effector cell population. Our studies provide a new rational for combination therapy of IL-15 and PD-1 antibody to overcome cancer resistance for patients who did not respond to initial PD-1 therapy. Citation Format: Henan Zhang, Xin Liu, Siyu Cao, Lingling Chen, Susan Harrington, Ying Li, Aaron Mansfield, Sean Park, Yiyi Yan, Roxana Dronca, Haidong Dong. Expansion of CX3CR1+ cytotoxic CD8+ T cells provides a rationale for IL-15 in combination cancer therapy with PD-1 antibody [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 4080.

Abstract 3208: Using T cell engineering plus triple checkpoint blockade to enhance the efficacy of adoptive immunotherapy in ovarian cancer

Abstract Over 20,000 women are diagnosed with ovarian cancer in the United States annually, and over half will die within 5 years. Outcomes have changed little in the last 20 years, highlighting the need for therapy innovation. One promising new strategy employs immune T cells engineered to target proteins uniquely overexpressed in tumors; such T cell immunotherapies have the potential to control tumor growth without toxicity to healthy tissues. In considering candidate immunotherapy targets, we focused on mesothelin (MSLN), which contributes to invasive progression and malignancy in ovarian cancer but has limited expression in healthy cells. We showed that T cells engineered to express a human or mouse MSLN-specific high-affinity T cell receptor (TCRMSLN) can kill human patient-derived ovarian cancer cell lines or the murine ID8 cell line, respectively. In a disseminated ID8 tumor model, adoptively transferred TCRMSLN T cells preferentially accumulated within established tumors, delayed ovarian tumor growth, and significantly prolonged mouse survival. However, our data also revealed that the ovarian tumor microenvironment (TME) limits engineered T cell persistence and cancer cell killing. To identify immunosuppressive features active in both the human and murine ovarian TME, we performed gene expression analyses. Deep transcriptome profiling confirmed similar gene expression signatures in human cancers and in the preclinical ID8 model. Among these, RNA sequencing detected PD-L1, Galectin-9 and Galectin-3, ligands for CD8 T cell-expressed PD-1, Tim-3 and Lag-3 ‘checkpoint’ receptors, respectively. We also measured PD-L1, Galectin-9 and Galectin-3 expression in human and mouse ovarian cancers by flow cytometry and immunohistochemistry, and multiplex immunohistochemistry of human ovarian tumors confirmed the presence of endogenous CD8 T cells expressing one, two or all three inhibitory receptors. Moreover, flow cytometry revealed that TCRMSLN-transduced T cells increase expression of the inhibitory receptors in ID8 tumors relative to cells in the spleen as early as three weeks after transfer, in association with decreased production of anti-tumor cytokines. Based on our results, we hypothesized that we could overcome engineered T cell suppression via inhibitory receptor ligation. We treated tumor-bearing mice with TCRMSLN T cells plus anti-PD-1, anti-Tim-3 and/or anti-Lag-3 checkpoint-blocking antibodies, targeting up to three inhibitory receptors simultaneously. Triple checkpoint blockade dramatically increased anti-tumor cytokine production by intratumoral TCRMSLN T cells. As many solid tumors both overexpress MSLN as well as PD-1, Tim-3 and Lag-3, the use of multi-checkpoint blockade with engineered T cells has real potential to also enhance the efficacy of engineered adoptive T cell therapy against other malignancies. Citation Format: Kristin G. Anderson, Madison G. Burnett, Valentin Voillet, Edison Y. Chiu, Breanna M. Bates, Nicolas M. Garcia, Raphael Gottado, Philip D. Greenberg. Using T cell engineering plus triple checkpoint blockade to enhance the efficacy of adoptive immunotherapy in ovarian cancer [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 3208.

Abstract 4085: DDR2 inhibition enhances response to anti-PD-1 immunotherapy

Abstract Therapies targeting PD-1 are used in multiple cancer types. While a fraction of patients show durable therapeutic responses, most remain unresponsive, highlighting an urgent need to better understand and improve these therapies. Using an in vivo screening approach with a customized shRNA pooled library, we identified DDR2 as a leading target for the enhancement of response to anti-PD-1 immunotherapy. Using isogenic in vivo murine models across five different tumor histologies, bladder, breast, colon, sarcoma and melanoma, we show that DDR2 depletion increases sensitivity to anti-PD-1 treatment compared to monotherapy. Combination treatment of tumor-bearing mice with anti-PD-1 and dasatinib, a tyrosine kinase inhibitor of DDR2, also led to tumor load reduction and in some cases, complete clearance. RNAseq and CyTOF analysis revealed higher CD8+ T cell populations in tumors with DDR2 depletion and those treated with dasatinib when either was combined with anti-PD-1 treatment. Our work provides strong scientific rationale for targeting DDR2 in combination with PD-1 inhibitors. Citation Format: Megan M. Tu, Francis Y. Lee, Robert T. Jones, Abigail K. Kimball, Elizabeth Saravia, Robert F. Graziano, Brianne Coleman, Krista Menard, Jun Yan, Erin Michaud, Han Chang, Hany A. Abdel-Hafiz, Andrii I. Rozhok, Jason E. Duex, Neeraj Agarwal, Ana Chauca-Diaz, Linda K. Johnson, Terry L. Ng, John C. Cambier, Eric T. Clambey, James C. Costello, Alan J. Korman, Dan Theodorescu. DDR2 inhibition enhances response to anti-PD-1 immunotherapy [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 4085.

Alternative splicing in human cancer cells is modulated by the amiloride derivative 3,5-diamino-6-chloro-N-(N-(2,6-dichlorobenzoyl)carbamimidoyl)pyrazine-2-carboxide.

Alternative splicing (AS) is a process that enables the generation of multiple protein isoforms with different biological properties from a single mRNA. Cancer cells often use the maneuverability conferred by AS to produce proteins that contribute to growth and survival. In our previous studies, we identified that amiloride modulates AS in cancer cells. However, the effective concentration of amiloride required to modulate AS is too high for use in cancer treatment. In this study, we used computational algorithms to screen potential amiloride derivatives for their ability to regulate AS in cancer cells. We found that 3,5‐diamino‐6‐chloro‐N‐(N‐(2,6‐dichlorobenzoyl)carbamimidoyl)pyrazine‐2‐carboxamide (BS008) can regulate AS of apoptotic gene transcripts, including HIPK3, SMAC, and BCL‐X, at a lower concentration than amiloride. This splicing regulation involved various splicing factors, and it was accompanied by a change in the phosphorylation state of serine/arginine‐rich proteins (SR proteins). RNA sequencing was performed to reveal that AS of many other apoptotic gene transcripts, such as AATF, ATM, AIFM1, NFKB1, and API5, was also modulated by BS008. In vivo experiments further indicated that treatment of tumor‐bearing mice with BS008 resulted in a marked decrease in tumor size. BS008 also had inhibitory effects in vitro, either alone or in a synergistic combination with the cytotoxic chemotherapeutic agents sorafenib and nilotinib. BS008 enabled sorafenib dose reduction without compromising antitumor activity. These findings suggest that BS008 may possess therapeutic potential for cancer treatment.

An Isoxazole Derivative SHU00238 Suppresses Colorectal Cancer Growth through miRNAs Regulation.

Colorectal cancer (CRC) is a leading cause of cancer-related deaths worldwide. Isoxazoline and isoxazole derivatives represent an important class of five-membered heterocycles, which play a pivotal role in drug discovery. In our previous study, we developed a series of isoxazole derivatives with an efficient method. In this study, we evaluated their effects on tumor cell growth. HCT116 cells were treated with isoxazole derivatives; an cholecystokinin octapeptide (CCK-8) assay was used to calculate the IC50 (half maximal inhibitory concentration) of each derivative. Compound SHU00238, which was obtained by the copper nitrate-mediated [2+2+1] cycloaddition reaction of olefinic azlactone with naphthalene-1,4-dione, has a lower IC50; we analyzed its inhibitory activity in further assays. Cell apoptosis was estimated by flow cytometry analysis in vitro. SHU00238 injection was used to treat tumor-bearing mice. We found that SHU00238 suppressed cell viability and promoted cell apoptosis in vitro. SHU00238 treatment significantly inhibited colonic tumor growth in vivo. Furthermore, we compared the miRNAs expression changes in HCT116 cells before and after SHU00238 treatment. MiRNA profiling revealed that SHU00238 treatment affected cell fate by regulating a set of miRNAs. In conclusion, SHU00238 impedes CRC tumor cell proliferation and promotes cell apoptosis by miRNAs regulation.

MRNA Delivery for Therapeutic Anti-HER2 Antibody Expression InVivo.

Antibody-based drugs are a leading class of biologics used to treat a variety of diseases, including cancer. However, wide antibody implementation is hindered by manufacturing challenges and high production cost. Use of in-vitro-transcribed mRNA (IVT-mRNA) for endogenous protein expression has the potential to circumvent many of the shortcomings of antibody production and therapeutic application. Here, we describe the development of an IVT-mRNA system for invivo delivery of a humanized anti-HER2 (also known as ERBB2) antibody, trastuzumab, and demonstrate its anticancer activity. We engineered the IVT-mRNA sequence to maximize expression, then formulated the IVT-mRNA into lipid-based nanoparticles (LNPs) to protect the mRNA from degradation and enable efficient invivo delivery. Systemic delivery of the optimized IVT-mRNA loaded into LNPs resulted in antibody serum concentrations of 45± 8.6μg/mL for 14days after LNP injection. Further studies demonstrated an improved pharmacokinetic profile of the produced protein compared to injection of trastuzumab protein. Finally, treatment of tumor-bearing mice with trastuzumab IVT-mRNA LNPs selectively reduced the volume of HER2-positive tumors and improved animal survival. Taken together, the results of our study demonstrate that using IVT-mRNA LNPs to express full-size therapeutic antibodies in the liver can provide an effective strategy for cancer treatment and offers an alternative to protein administration.

Macrophage migration inhibitory factor (MIF):A novel therapeutic target against aggressive breast cancer

Triple negative breast cancer (TNBC), defined as loss of estrogen, progesterone and Her2 receptors, is a subtype of breast cancer with worse prognosis and poor survival rate. Increased inflammation further reduces the TNBC patient survival. Macrophage migration inhibitory factor (MIF) is a pro‐inflammatory molecule known to enhance breast cancer progression and metastasis. However, not much is known about the role of MIF in TNBC progression and metastasis. MIF interacts with various oncogenic molecules and signaling pathways. Therefore, targeting MIF might be a promising strategy to inhibit TNBC progression and metastasis. Here we report that CPSI‐1306, a small molecule inhibitor specific to MIF, possesses the ability to target TNBC cell in vitro by inducing apoptosis. The mechanistic studies revealed that CPSI‐1306 induces apoptosis by enhancing reactive oxygen species (ROS), mitochondria membrane potential alteration, cytochrome C release and activation of Caspases. In addition, CPSI‐1306 also inhibits cell survival and apoptosis signaling molecules, including AKT, PDK and RAF expression. Next, we analyzed the clinical utility of CPSI‐1306 using mouse MVT‐1 cells derived orthotopic and human MDA‐MB‐231 cells derived xenograft TNBC mouse models. The oral treatment of tumor bearing mice with CPSI‐1306 significantly inhibited TNBC growth and metastasis to the lungs in a dose dependent manner. The histological analysis of tumors showed higher number of apoptotic cells in CPSI‐1306 treated tumors compared to vehicle controls. We also observed an association of higher MIF mRNA expression with poor patient survival, including TNBC, by using publically available datasets. These results suggest that CPSI‐1306 induces cell death in TNBC cells via activating ROS and mitochondria‐mediated apoptosis and significantly inhibits TNBC growth and metastasis in pre‐clinical TNBC mouse models.Support or Funding InformationDOD, Breakthrough AwardThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Targeting EZH2 for glioma therapy with a novel nanoparticle-siRNA complex.

BackgroundFor the past few years, gene-therapy has recently shown considerable clinical benefit in cancer therapy, and the applications of gene therapies in cancer treatments continue to increase perennially. EZH2, an ideal candidate for tumor gene therapy, plays an important role in the tumorigenesis.MethodsIn this study, we developed a novel gene delivery system with a self-assembly method by Methoxy polyethylene glycol-polycaprolactone (MPEG-PCL) and DOTAP(DMC). And EZH2si-DMC was used to research anti-glioma both in vitro and in vivo.ResultsDMC with zeta-potential value of 36.7 mV and size of 35.6 nm showed good performance in the delivery siRNA to glioma cell in vitro with high 98% transfection efficiency. EZH2si-DMC showed good anti-glioma effect in vitro through inducing cell apoptosis and inhibiting cell growth. What’s more, treatment of tumor-bearing mice with DMC-EZH2si complex had significantly inhibited tumor growth at the subcutaneous model in vivo by inhibiting EZH2 protein expression, promoting apoptosis and reducing proliferation.ConclusionThe EZH2 siRNA and DMC complex may be used to treat the glioma in clinical as a new drug.

Anti-PD-L1 Treatment Results in Functional Remodeling of the Macrophage Compartment.

Checkpoint inhibitors like anti-PD1/PD-L1 have demonstrated significant therapeutic efficacy in a subset of patients partly through reinvigoration of CD8 T cells. However, their impact on myeloid cells remains largely unknown. Here, we report that anti-PD-L1 treatment favorably impacts the phenotype and function of tumor macrophages by polarizing the macrophage compartment toward a more proinflammatory phenotype. This phenotype was characterized by a decrease in Arginase-I (ARG1) expression and an increase in iNOS, MHCII, and CD40 expression. Whole-transcriptome profiling further confirmed extensive polarization of both tumor monocytes and macrophages from a suppressive to a proinflammatory, immunostimulatory phenotype. This polarization was driven mainly through IFNγ and was associated with enhanced T-cell activity. Transfer of monocytes into anti-PD-L1-treated tumor-bearing mice led to macrophage differentiation into a more proinflammatory phenotype, with an increase in CD8 T cells expressing granzyme-B and an increase in the CD8/Treg ratio compared with control-treated mice. Although in responsive tumor models, anti-PD-L1 treatment remodeled the macrophage compartment with beneficial effects on T cells, both macrophage reprogramming and depletion were needed to maximize anti-PD-L1 responses in a tumor immune contexture with high macrophage burden. Our results demonstrate that anti-PD-L1 treatment can favorably remodel the macrophage compartment in responsive tumor models toward a more proinflammatory phenotype, mainly through increased IFNγ levels. They also suggest that directly targeting these cells with reprogramming and depleting agents may further augment the breadth and depth of response to anti-PD-L1 treatment in less responsive or more macrophage-dense tumor microenvironments. SIGNIFICANCE: This work demonstrates that increased IFNγ signaling following anti-PD-L1 treatment can remodel the macrophage compartment to enhance T-cell responses.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/7/1493/F1.large.jpg.

Tumor targeting and microenvironment-responsive multifunctional fusion protein for pro-apoptotic peptide delivery.

The great therapeutic potential of peptides has not yet been achieved, mainly due to their remarkably short in vivo half-life. Although conjugation to macromolecules has been an effective way of improving protein in vivo half-life, the steric hindrance of macromolecules usually reduces the in vivo efficacy of peptides. Here we report a complex delivery system made from PsTag polypeptide, polyglutamic acid chain, matrix metalloproteinase 2 (MMP2)-degradable domain and cationic cell penetrating peptide for anticancer peptide delivery. Clear evidence was shown in vitro and in vivo to demonstrate that this multifunctional protein fusing a pro-apoptotic KLAKLAKKLAKLAK (KLA), named PAK, can increase circulation time in blood, enhance accumulation at tumor sites, eliminate the PsTag domain and the polyanionic sequence when triggered by tumor overexpressing MMP2, and then expose the cell penetrating peptide to realize the potent cellular uptake of KLA. Treatment of tumor-bearing mice with PAK could markedly induce tumor cells apoptosis and inhibit tumor growth, with no significant adverse effects. These results suggest our fusion protein can be a potential delivery system for peptide delivery in cancer treatments.

NIR-Triggered Phototherapy and Immunotherapy via an Antigen-Capturing Nanoplatform for Metastatic Cancer Treatment.

Combined phototherapy and immunotherapy demonstrates strong potential in the treatment of metastatic cancers. An upconversion nanoparticle (UCNP) based antigen‐capturing nanoplatform is designed to synergize phototherapies and immunotherapy. In particular, this nanoplatform is constructed via self‐assembly of DSPE‐PEG‐maleimide and indocyanine green (ICG) onto UCNPs, followed by loading of the photosensitizer rose bengal (RB). ICG significantly enhances the RB‐based photodynamic therapy efficiency of UCNP/ICG/RB‐mal upon activation by a near‐infrared (NIR) laser, simultaneously achieving selective photothermal therapy. Most importantly, tumor‐derived protein antigens, arising from phototherapy‐treated tumor cells, can be captured and retained in situ, due to the functionality of maleimide, which further enhance the tumor antigen uptake and presentation by antigen‐presenting cells. The synergized photothermal, photodynamic, and immunological effects using light‐activated UCNP/ICG/RB‐mal induces a tumor‐specific immune response. In the experiments, intratumoral administration of UCNP/ICG/RB‐mal, followed by noninvasive irradiation with an NIR laser, destroys primary tumors and inhibits untreated distant tumors, using a poorly immunogenic, highly metastatic 4T1 mammary tumor model. With the simultaneous use of anti‐CTLA‐4, about 84% of the treated tumor‐bearing mice achieve long‐term survival and 34% of mice develop tumor‐specific immunity. Overall, this antigen‐capturing nanoplatform provides a promising approach for the treatment of metastatic cancers.