Tumor-bearing Mice Research Articles

A high-fat diet promotes cancer progression by inducing gut microbiota–mediated leucine production and PMN-MDSC differentiation

A high-fat diet (HFD) is a high-risk factor for the malignant progression of cancers through the disruption of the intestinal microbiota. However, the role of the HFD-related gut microbiota in cancer development remains unclear. This study found that obesity and obesity-related gut microbiota were associated with poor prognosis and advanced clinicopathological status in female patients with breast cancer. To investigate the impact of HFD-associated gut microbiota on cancer progression, we established various models, including HFD feeding, fecal microbiota transplantation, antibiotic feeding, and bacterial gavage, in tumor-bearing mice. HFD-related microbiota promotes cancer progression by generating polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs). Mechanistically, the HFD microbiota released abundant leucine, which activated the mTORC1 signaling pathway in myeloid progenitors for PMN-MDSC differentiation. Clinically, the elevated leucine level in the peripheral blood induced by the HFD microbiota was correlated with abundant tumoral PMN-MDSC infiltration and poor clinical outcomes in female patients with breast cancer. These findings revealed that the "gut-bone marrow-tumor" axis is involved in HFD-mediated cancer progression and opens a broad avenue for anticancer therapeutic strategies by targeting the aberrant metabolism of the gut microbiota.

Yogurt Alleviates Cyclophosphamide-Induced Immunosuppression in Mice through D-Lactate.

Numerous studies have investigated the immunomodulatory effects of yogurt, but the underlying mechanism remained elusive. This study aimed to elucidate the alleviating properties of yogurt on immunosuppression and proposed the underlying mechanism was related to the metabolite D-lactate. In the healthy mice, we validated the safety of daily yogurt consumption (600 μL) or D-lactate (300 mg/kg). In immunosuppressed mice induced by cyclophosphamide (CTX), we evaluated the immune regulation of yogurt and D-lactate. The result showed that yogurt restored body weight, boosted immune organ index, repaired splenic tissue, recovered the severity of delayed-type hypersensitivity reactions and increased serum cytokines (IgA, IgG, IL-6, IFN-γ). Additionally, yogurt enhanced intestinal immune function by restoring the intestinal barrier and upregulating the abundance of Bifidobacterium and Lactobacillus. Further studies showed that D-lactate alleviated immunosuppression in mice mainly by promoting cellular immunity. D-lactate recovered body weight and organ development, elevated serum cytokines (IgA, IgG, IL-6, IFN-γ), enhanced splenic lymphocyte proliferation and increased the mRNA level of T-bet in splenic lymphocyte to bolster Th1 differentiation. Finally, CTX is a chemotherapeutic drug, thus, the application of yogurt and D-lactate in the tumor-bearing mouse model was initially explored. The results showed that both yogurt (600 μL) and D-lactate (300 mg/kg) reduced cyclophosphamide-induced immunosuppression without promoting tumor growth. Overall, this study evaluated the safety, immune efficacy and applicability of yogurt and D-lactate in regulating immunosuppression. It emphasized the potential of yogurt as a functional food for immune regulation, with D-lactate playing a crucial role in its immunomodulatory effects.

Folate-PEG-PROTAC Micelles for Enhancing Tumor-Specific Targeting Proteolysis In Vivo.

Proteolysis targeting chimeras (PROTACs) technology is rapidly developed as a novel and selective medicinal strategy for the degradation of cellular proteins in cancer therapy. However, the applications of PROTACs as heterobifunctional molecules are largely limited by high molecular weight, low bioavailability, poor permeability, insufficient targeting, and low efficacy in vivo. Herein, self-assembling micelles of FA-PEG-PROTAC are designed for cancer cell selective targeting and reductive-response proteolysis in tumor-bearing mice. FA-PEG-PROTAC is prepared by conjugating folic acid (FA)-PEG with EGFR-targeting PROTAC via a disulfide bond. The FA-PEG-PROTAC micelles, formed by self-assembling, are demonstrated to significantly improve tumor targeting efficacy and exhibit excellent anti-tumor efficacy in the mouse xenograft model compared to the traditional PROTACs. The strategy of applying self-assembled FA-PEG-PROTAC micelles in tumor therapy can not only improve targeted proteolysis efficiency but also broaden applications in the development of PROTAC-based drugs.

Combination therapy with oncolytic virus and T cells or mRNA vaccine amplifies antitumor effects

Antitumor therapies based on adoptively transferred T cells or oncolytic viruses have made significant progress in recent years, but the limited efficiency of their infiltration into solid tumors makes it difficult to achieve desired antitumor effects when used alone. In this study, an oncolytic virus (rVSV-LCMVG) that is not prone to induce virus-neutralizing antibodies was designed and combined with adoptively transferred T cells. By transforming the immunosuppressive tumor microenvironment into an immunosensitive one, in B16 tumor-bearing mice, combination therapy showed superior antitumor effects than monotherapy. This occurred whether the OV was administered intratumorally or intravenously. Combination therapy significantly increased cytokine and chemokine levels within tumors and recruited CD8+ T cells to the TME to trigger antitumor immune responses. Pretreatment with adoptively transferred T cells and subsequent oncolytic virotherapy sensitizes refractory tumors by boosting T-cell recruitment, down-regulating the expression of PD-1, and restoring effector T-cell function. To offer a combination therapy with greater translational value, mRNA vaccines were introduced to induce tumor-specific T cells instead of adoptively transferred T cells. The combination of OVs and mRNA vaccine also displays a significant reduction in tumor burden and prolonged survival. This study proposed a rational combination therapy of OVs with adoptive T-cell transfer or mRNA vaccines encoding tumor-associated antigens, in terms of synergistic efficacy and mechanism.

CCR5 antagonist maraviroc alleviates doxorubicin-induced neuroinflammation and neurobehavioral deficiency by regulating NF-κB/NLRP3 signaling in a breast cancer mouse model

The chemotherapeutic agent Doxorubicin (DOX) is known to cause chemotherapy-induced cognitive impairment (CICI). Maraviroc, a potent C–C chemokine receptor 5 (CCR5) antagonist, shows neuroprotective properties, while its role in CICI remains unclear. This study determined the therapeutic potential of maraviroc on CICI. Adult C57BL/6J mice with implanted breast cancer cells received four weekly intraperitoneal injections of saline (Control group), 5 mg/kg DOX (DOX group), 10 mg/kg maraviroc (MVC group), or 5 mg/kg DOX with 10 mg/kg maraviroc (DOX + MVC group). The Morris Water Maze (MWM) was used for neurobehavioural test. Western blot analysis and immunofluorescence were used to evaluate the expressions of inflammatory markers, apoptosis-related proteins, and synaptic-related proteins. The volume and weight of tumor were also evaluated after treatments. DOX treatment significantly increased chemokines (CCL3, CCL4) and inflammatory cytokines (IL-1β, TNF-α) in tumor-bearing mice hippocampus. While maraviroc administration reduced hippocampal proinflammatory factors compared to the DOX group. Furthermore, it also lowered apoptosis markers, restored synaptic proteins levels, and inhibited the NF-κB/NLRP3 pathway. Accordingly, maraviroc treatment significantly improved DOX-induced neurobehavioural impairments as evidenced by an increased number of platform crossings and percentage of target quadrant time in the MWM test. Additionally, when combined with DOX, maraviroc had additional inhibitory effects on tumor growth. These findings suggest that maraviroc can mitigate DOX-induced CICI by suppressing elevated proinflammatory chemokines and cytokines through the NF-κB/NLRP3 pathway, potentially offering an anti-tumor benefit. This research presents a promising therapeutic approach for DOX-induced CICI, enhancing the safety and efficacy of cancer treatments.

Abstract PO1-25-08: Evaluating the role of CDK4/6 inhibition on STAT3 activation in a transgenic mouse model of HER2-positive breast cancer

Abstract Background: The human epidermal growth factor receptor 2 (HER2) is a receptor tyrosine kinase overexpressed in approximately 20-25% of breast cancers with varying biological differences according to hormone receptor (HR) positive status. Combination of chemotherapy with dual HER2 blockade and endocrine therapy (adjuvant) or CDK4/6 inhibitor (CDK4/6i) therapy (metastatic) is standard of care for HER2-positive/HR-positive breast cancer. However, de-escalation of chemotherapy remains of significant interest given associated toxicities and, as a result, alternative therapeutic strategies are needed. One such strategy is targeting the signal transducer and activator of transcription 3 (STAT3) which is constitutively activated in the HER-2 positive setting. Moreover, there is preclinical evidence to suggest that phosphorylated STAT3 (pSTAT3) induces overexpression of cyclin D1 (CCND1), which along with CDK4 may mediate resistance to targeted therapy for HER2-positive breast cancer. However, it is unclear if pSTAT3 plays a significant role in tumorigenesis or the development of resistance to therapy in HER2-positive disease. We aim to evaluate the effect of CDK4/6i therapy with palbociclib on tumor growth in an inducible transgenic HER2-positive mouse model and the impact on pSTAT3 and cyclin D1 levels. Methods: Using an inducible transgenic mouse mammary tumor virus (MMTV) model of HER2-positive breast cancer, we evaluated STAT3 phosphorylation in tumors driven by HER2 expression in addition to the impact of CDK4/6 inhibition with palbociclib on tumorigenesis and pSTAT3 activation. To model tumor recurrence, doxycycline was withdrawn from tumor bearing mice and tumor regression was observed. Subsequently, the mice developed recurrent mammary tumors. Given recurrent tumors in this model are driven by a HER2-independent mechanism, we examined expression of cell cycle proteins together with STAT3 phosphorylation to determine association with recurrence. Result: In addition to expressing HER2, the primary tumors expressed estrogen receptor (ERα), Rb, cyclin D1, and CDK4/6 proteins. STAT3 phosphorylation was also observed. Treatment with palbociclib decreased Rb phosphorylation and decreased tumor growth compared to vehicle treated mice (%TGI 79.37% after 21 days). Furthermore, pSTAT3 levels were not altered with palbociclib therapy. When recurrent tumors were analyzed, they lacked HER2 expression but expressed cyclin D1 and CDK4/6 supporting that tumor growth is driven by cyclin D1/CDK4 pathway. Recurrent tumors also demonstrated pSTAT3 despite lack of HER2 expression suggesting that STAT3 phosphorylation in the recurrent tumors is mediated by an alternative mechanism, potentially the CDK4/6 pathway. Conclusion: We demonstrate STAT3 activation in a mouse model of HER2 driven breast cancer and show persistent activation in recurrent tumors lacking HER2 expression rendering them resistant to HER2-targeted therapy. We also demonstrate the anti-tumor effect of palbociclib in primary tumors with HER2-dependent proliferation. Taken together, our findings suggest that pSTAT3 is a viable therapeutic target and provides the rationale for combination therapy with CDK4/6 inhibition using palbociclib and STAT3 inhibition with a novel STAT3 inhibitor in HER2-positive breast cancer. This therapeutic approach may represent a potential chemotherapy de-escalation strategy. Citation Format: Taiwo Adesoye, Linjie Luo, Tuyen Bui, Serena Kim, Hannah Wingate, Debu Tripathy, Kelly Hunt, Khandan Keyomarsi. Evaluating the role of CDK4/6 inhibition on STAT3 activation in a transgenic mouse model of HER2-positive breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-25-08.

Abstract RF02-02: BBO-10203, a first-in-class, orally bioavailable, selective covalent small molecule that inhibits RAS-driven PI3Kalpha activity without affecting glucose metabolism

Abstract PI3Kα is the most mutated kinase and the second most mutated oncogene in human cancer. Activation of PI3Ka can be achieved by receptor tyrosine kinases such as insulin receptor and insulin-like growth factor receptor 1 and/or by directly interacting with RAS family members. Previous elegant preclinical studies have established that RAS-driven PI3Ka activation is important in tumor cells but may not be involved in cell types controlling glucose metabolism. Alpelisib, a small molecule inhibitor of the kinase activity of PI3Ka, has been approved for the treatment of ER+ PIK3CA mutant breast cancer in combination with fulvestrant after endocrine therapy in advanced or metastatic breast cancer based on an improvement in PFS versus fulvestrant alone. Inhibition of PI3Ka activity by alpelisib in normal tissues resulted in a severe (G3/4) hyperglycemia rate of 37% with frequent dose interruptions and discontinuations. Additionally, preclinical studies have demonstrated that the dysregulation of glucose homeostasis resulting from PI3Ka kinase inhibition leads to hyperinsulinemia that increases pathway flux, rendering kinase inhibitors less effective. Here, we report on a novel covalent small molecule designed to inhibit RAS-mediated activation of the AKT pathway via PI3Ka without the resultant hyperglycemia associated with direct inhibition of PI3Ka kinase activity. BBO-10203 disrupts the physical interaction between RAS and PI3Ka in tumor cells resulting in potent signaling pathway inhibition. This agent selectively binds to PI3Ka and disrupts its interaction with K-,H-, and N-RAS with low single digit nanomolar potency (~5 nM). Breaking the interaction between these two oncogenes inhibits basal pAKT cellular levels (BT-474/KYSE-410 IC50: ~5 nM) in HER2 amplified (HER2amp) and wild-type or mutant PI3Ka cell lines. Even though BBO-10203 does not inhibit the kinase activity of PI3Ka, its effects on cancer cell signaling inhibition and transcriptional regulation highly resemble those of alpelisib. BBO-10203 displays excellent drug-like properties and oral bioavailability. Single dose treatment of KYSE-410 (HER2amp/KRASG12C) tumor bearing mice with increasing doses (1-100 mg/kg, PO) of BBO-10203 results in dose and time dependent inhibition of pAKT in vivo. Maximal inhibition (~80%) is achieved at 30 mg/kg and lasts for 24 hours. Repeated dose treatment of tumor bearing mice with BBO-10203 is well tolerated and results in significant efficacy in PIK3CA mutant as well as HER2amp human xenograft models. In the KYSE-410 xenograft model, BBO-10203 daily oral dosing of 30 mg/kg results in significant tumor regressions. Importantly, treatment with BBO-10203 does not affect insulin signaling in differentiated adipocytes in vitro, nor does it impact glucose metabolism in vivo at 3-times the maximal efficacious dose level in xenograft studies. In conclusion, we have identified a novel approach to inhibit the PI3Ka signaling pathway by blocking its interaction with, and activation by RAS. This approach can achieve strong pAKT inhibition in tumor cells without changes in glucose metabolism. Clinical investigation of BBO-10203 for the treatment of both ER+/PIK3CA mutant and HER2amp breast cancer is warranted. Citation Format: Pedro Beltran, Simanshu Dhirendra, Rui Xu, Ming Chen, Daniel Czyzyk, Sofia Donovan, Siyu Feng, Cindy Feng, Lijuan Fu, Felice Lightstone, Ken Lin, Anna Maciag, Dwight Nissley, Erin Riegler, Kerstin Sinkevicius, Andrew Stephen, James Stice, David Turner, Bin Wang, Keshi Wang, Yue Yang, Cathy Zhang, Frank McCormick, Eli Wallace. BBO-10203, a first-in-class, orally bioavailable, selective covalent small molecule that inhibits RAS-driven PI3Kalpha activity without affecting glucose metabolism [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr RF02-02.

Abstract PO3-18-04: MBRC-101: a novel antibody-drug conjugate (ADC) targeting the membrane-associated tyrosine kinase receptor EphA5 in breast cancer

Abstract Objectives: ADCs have emerged as a major advance in contemporary medical oncology. The receptor tyrosine kinase (RTK) EphA5 possesses the classical receptor kinase topology with an extracellular-binding domain, a single-pass transmembrane domain, and a cytoplasmic kinase domain. We have previously shown that EphA5 is highly expressed in non-small cell lung cancer and contributes to DNA-damage repair and radiation therapy resistance. Others have reported EphA5 expression in gastric, ovarian, and pancreatic cancers. Here, we investigated the expression of EphA5 in breast cancer (BC) and assessed EphA5 as a potential cancer-specific target using a novel ADC designated MBRC-101. Methods: MBRC-101 is composed of a humanized anti-EphA5 IgG1 kappa monoclonal antibody conjugated to the small molecule microtubule-disrupting agent monomethyl auristatin E (MMAE) via a protease cleavable valine-citrulline (vc) ThioBridge linker (ThioBridge™-Glu-[Val-Cit-PAB-MMAE] PEG [24u]). MBRC-101 averages a drug-to-antibody ratio (DAR)=4. The specificity profile of the anti-EphA5 monoclonal antibody was characterized using a Membrane Proteome Array (MPA). Receptor-mediated antibody internalization was determined by real-time live-cell analysis; cell killing assays used both live-cell analysis of cell confluence and the CellTiter-Glo® luminescent cell viability assay. Expression of EphA5 in archival human breast tumor tissue sections was evaluated by immunohistochemistry (IHC) using a commercial polyclonal antibody against human EphA5. In vivo efficacy studies used patient-derived xenograft (PDX) models of BC. Results: IHC demonstrated robust and selective EphA5 expression in 87% (20 of 23) of triple negative breast cancer (TNBC) and 88% (23 of 26) of hormone receptor-positive (HR+) patient tumors tested. EphA5 expression was not detected in adjacent, non-malignant breast tissue. Antibody-specificity profile testing showed that the anti-EphA5 antibody bound exclusively to EphA5 and did not cross-react with other members of the Eph receptor/ephrin ligand family. Binding of the anti-EphA5 antibody to EphA5 on the cell surface triggered rapid internalization and processing of the EphA5/antibody complex through the endosomal-lysosomal system. Consistently, MBRC-101 was cytotoxic to EphA5-expressing cells and its activity was concentration-dependent and commensurate to the levels of EphA5 on the cell surface. In PDX murine models of TNBC, once weekly administrations of intravenous (IV) MBRC-101 showed dose-dependent, robust, and reproducible anti-tumor activity in vivo. Partial tumor responses were observed at doses of 2.5 mg/Kg IV and complete and nearly complete tumor responses starting at 5 mg/Kg IV. Doses up to 10 mg/Kg IV were well-tolerated in tumor-bearing mice with no observable weight loss. Pre-clinical safety confirmed MBRC-101 is also well tolerated in rats and in cynomolgus monkeys. Conclusions: These results support EphA5 as a new and promising membrane-associated molecular target for the design and development of ADC-based therapies against hormone-positive breast cancer and TNBC and support the conduct of a Phase 1/1b study of MBRC-101 in patients with breast cancer. Citation Format: Fernanda Staquicini, Fenny Tang, Vanessa de Oliveira, Daniela Staquicini, Yongjian Wu, Kirstin Barnhart, J Kellogg Parsons, Wadih Arap, Isan Chen, Renata Pasqualini. MBRC-101: a novel antibody-drug conjugate (ADC) targeting the membrane-associated tyrosine kinase receptor EphA5 in breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-18-04.

Green Synthesis of Gold Nanoparticles from Polygahatous Polysaccharides and Their Anticancer Effect on Hepatic Carcinoma through Immunoregulation

Green Synthesis of Gold Nanoparticles from Polygahatous Polysaccharides and Their Anticancer Effect on Hepatic Carcinoma through Immunoregulation

Abstract PO2-25-07: PEGylated Functional Upstream Domain (PEG-FUD): an anti-cancer therapy for breast Cancer

Abstract Background: Breast cancer (BC) is the most common cancer diagnosed in women. In breast cancers, the extracellular matrix (ECM) is known to play a key role in disease progression by mediating cell signaling processes that drive cancer cell proliferation, migration and invasion. Work by our group identified that aligned collagen fibers perpendicular to the tumor boundary are prognostic of poor patient prognosis. Further, through matrix targeted proteomics we determined that the ECM protein, Fibronectin (FN), organizes with aligned collagen fibers in BC tissues. FN is known as the master regulator of ECM assembly because FN deposition precedes and regulates the deposition of several other ECM proteins. Clinical evidence shows that high expression of FN levels in BC patients correlates with an increased mortality risk. Due to the abundant expression of FN in the tumor microenvironment, FN has been a useful biomarker for cancer imaging and therapy. Despite multiple efforts in developing therapies that target the ECM, currently there are no effective ECM treatments available due to toxicity and lack of specificity. Methods: To target abnormal FN deposition, we used a FN binding peptide, Functional Upstream Domain (FUD), derived from the F1 adhesin from Streptococcus pyogenes. PEGylated-FUD (PEG-FUD) is a potent inhibitor of FN assembly which binds the 70 kDa N-terminal region of FN with high affinity. We hypothesize that PEG-FUD will localize to the tumor site and that targeted disruption of FN assembly with PEG-FUD will reduce ECM deposition, thus block tumor progression in-vivo. In this study, we used an in vivo imaging system to assess the biodistribution of 20-kDa PEG-FUD following subcutaneous injection of Cy5 labeled peptide in 4T1 mammary tumor bearing mice. Additionally, in a second therapeutic experiment, we treated 4T1 mammary tumor bearing mice with 20-kDa PEG-FUD every 48 hr for a total of 10 treatments and measured tumor volume as an indicator of primary tumor burden. Results: As anticipated, we observed PEG-FUD’s accumulation and localization in 4T1 tumors. Additionally, PEG-FUD treatment significantly reduced tumor growth compared to saline treatment. There were no observed changes in standard toxicity assessments such as body weight and spleen weight among treatment groups. After PEG-FUD’s therapeutic treatment, we observed a significant reduction in the deposition of FN matrix and an increase in cleaved caspase-3, a known marker for apoptosis by western blotting providing a potential mechanism of action of PEG-FUD inducing changes in tumor growth. Conclusions: PEG-FUD’s localization in tumors suggests its utility as a solo cancer imaging agent while providing indications that PEG-FUD can be used to deliver other therapeutics to the tumor site as a conjugate in the future. In addition, therapeutic treatment of PEG-FUD inhibited tumor growth providing preliminary evidence for PEG-FUD’s use as a tumor targeting anti-cancer agent. Citation Format: Metti Gari. PEGylated Functional Upstream Domain (PEG-FUD): an anti-cancer therapy for breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-25-07.

Abstract PO4-06-11: Development of fully human anti-progranulin monoclonal antibodies as inhibitors of triple negative breast cancer cell growth and tumor formation

Abstract Triple negative breast cancer (TNBC) is characterized by invasiveness and poor survival. Identifying novel TNBC targeted therapies to potentiate standard of care (SOC) therapy, is an unmet need. In the past 4 years, several targeted therapies for TNBC have been approved by the Food and Drug Administration (FDA). However, the identification of targeted therapies against other biological drivers of TNBC remains important. Progranulin (PGRN/GP88) is a biological driver of tumorigenesis, survival, and drug resistance in breast cancer. PGRN/GP88 tissue expression is an independent prognostic factor of recurrence while elevated serum PGRN/GP88 level is associated with poor outcomes such as progression of disease and shortened survival. PGRN/GP88 expression is elevated in 30% TNBC. The importance of inhibiting PGRN/GP88 effect on the proliferation and tumor growth of triple negative breast cancer cells was previously established. In the current study, in association with Precision Antibody, we developed anti-PGRN/GP88 fully human monoclonal antibodies by immunizing TC transgenic humanized mice with recombinant human progranulin. Within 60 days since the start of immunization, several fully human monoclonal antibodies were successfully developed, characterized by several functional assays including neutralization, inhibition of PGRN/GP88 cell surface binding, proliferation, migration in MDA-MB-231 cells in vitro and in vivo. Results: The inhibition of PGRN/GP88 action by fully human monoclonal antibodies inhibited proliferation and migration in a dose-and time-dependent fashion in TNBC cells. Octet analysis determined Kd in the range of 10−11 M to 10−10 M. Transwell assay showed that anti-PGRN treatment inhibited migration In vivo xenograft studies with MDA-MB-231 cells injected in athymic nude mice showed that several fully human anti-PGRN antibodies inhibited tumor growth when compared to antibody control treated mice. In vivo dose response of AG01 in MDAMB-231 tumor bearing mice will be provided. Conclusion: PGRN/GP88 represents a therapeutic target for TNBC with two companion diagnostics (tissue test and ELISA to measure GP88 circulating levels). The use of the TC transgenic mice allows for the rapid development of fully human high affinity monoclonal antibodies bypassing the need for chimerization or humanization of mouse monoclonal antibodies. The development of fully human anti-progranulin antibodies provides novel targeted therapeutic option for TNBC. This work is supported by a an SBIR grant 5R44 CA 224718 from the National Cancer Institute to GS. Citation Format: Ginette Serrero, Jianping Dong, Jun Hayashi, Chun Dong. Development of fully human anti-progranulin monoclonal antibodies as inhibitors of triple negative breast cancer cell growth and tumor formation [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-06-11.

Abstract PO4-18-03: Combined Treatment with Pyrotinib and Chrysin Synergistically Improve the Autophagy Level in HER2-Positive Breast Cancer by Regulating the miR-16-5p/ZBTB16/G6PD Axis

Abstract Human epidermal growth factor receptor 2 (HER2)-positive breast cancer (BC) has been the most challenging subtype of BC, which consists of 20% of BC with an apparent correlation with poor prognosis. Despite that pyrotinib, a new HER2 inhibitor, has led to dramatic improvements in prognosis outcome, the efficacy of pyrotinib as monotherapy remain largely restricted due to its acquired resistance. Therefore, we aim at identifying a potent antitumor drug incorporated with pyrotinib for amplifying therapeutic efficacy for treating HER2-positive BC. Here, we reported a novel incorporation of pyrotinib in combination with chrysin, and explored its antitumor efficacy and the underlying mechanisms on HER2+ breast cancer. We determined that pyrotinib combined with chrysin yielded a potent synergistic effect to induce apoptosis and inhibit BT-474 and SK-BR-3 tumor cells, and suppressed in vivo tumor growth in tumor-bearing mice models. This may be mechanistically attributed to the induction of enhanced endoplasmic reticulum stress to increase the autophagy level. Furthermore, it was demonstrated that the combined treatment with pyrotinib and chrysin induced ubiquitination and G6PD degradation by regulating zinc finger and BTB/POZ domain-containing family protein 16 (ZBTB16) in tumorigenesis of BC. Besides, we identified that miR-16-5p is a potential upstream regulatory target of ZBTB16. Blocking miR-16-5p overexpression could inhibit HER2-positive tumorigenesis and significantly potentiate the efficacy of pyrotinib in combination with chrysin. Together, these findings demonstrate the utility of combined treatment with pyrotinib and chrysin as a potential option in the target treatment of HER2-positive BC through an unrecognized miR-16-5p/ZBTB16/G6PD axis. The miR-16-5p/ZBTB16/G6PD axis plays a crucial role in the pyrotinib plus chrysin-enabled anti HER2-positive BC Fig.1 a Cell viability of SK-BR-3 cells received various treatments. b Detection of cell cycle arrest of SK-BR-3 cells after various treatments. c Schematic diagram of the established protocol of the animal models. Photograph of resected tumor tissues, tumor volume (d), and tumor weight (e) of mice in different treatment groups during the whole testing period. f H&E, Ki67, and Tunel immunohistochemical staining of tumor sections of mice in various treatment groups. g Autophagy flux of SK-BR-3 cells labeled with mRFP-GFP-LC3 in the different treatment groups. h Expression level of ER stress markers in SK-BR-3 cells after various treatments via RT-qPCR and western blot. *P <0.05, compared with control group (DMSO), #P <0.05, compared with chrysin group, & P<0.05, compared with pyrotinib group. i Western blot analysis of the expression of G6PD in different treatment groups. j Fluorescence images of SK-BR-3 cells subjected to G6PD overexpression for the detection of autophagy level. k Ubibrowser database predicting the E3 ubiquitin ligases that may be involved in the regulation of G6PD ubiquitination. l Western blot analysis of G6PD protein half-life in SK-BR-3 cells with ZBTB16 silence. Cells were co-incubated with cycloheximide (CHX, 50μg/ml) for the indicated time. m Determination of the ubiquitination of G6PD in cells pretreated with 10 μM MG-132 for 3 h. Cells were transfected with ubiquitin after different treatments. The ubiquitinated G6PD was subjected to immunoprecipitation before western blot with ubiquitin antibody. n Dual-luciferase report verifying the targeting of ZBTB16 and miR-16-5p. o Transmission electron microscopy images of autophagosomes of tumors in various treatment groups (× 20000). p Immunohistochemical staining images of tumor slices for the determination of G6PD. q Schematic diagram for the underlying mechanism of pyrotinib combined chrysin against HER2-positive BC. Citation Format: Ting Luo, Xiaorong Zhong, Ping He, Dan Zheng, Yan Cheng, Kunrui Zhu. Combined Treatment with Pyrotinib and Chrysin Synergistically Improve the Autophagy Level in HER2-Positive Breast Cancer by Regulating the miR-16-5p/ZBTB16/G6PD Axis [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-18-03.

Abstract PO1-06-11: Exosomal HMGB1 induce PD-L1+-tumor associated macrophages through glycometabolic reprogramming to promote lung-tropic metastasis of triple negative breast cancer

Abstract Background: Lung metastasis occurs in 30% of triple negative breast cancer (TNBC), the detailed molecular mechanism remains largely unexplored. Accumulating evidence suggests that PD-L1+-tumor associated macrophages (PD-L1+-TAMs) are closely related to immune suppression, invasion, and metastasis. Tumor-derived exosomes have been reported to remodel tumor microenvironment and accelerate metastasis via packing and delivering a variety of biologically active molecules. Our previous studies found that exosomes from breast cancer cells polarized macrophages toward TAMs within the primary tumor nest. The aim of this study was to reveal whether TNBC-derived exosomes were able to polarize lung macrophages towards PD-L1+-TAMs and generate a pre-metastatic immunosuppressive niche, therefore discovering the underlying mechanism of lung-tropic metastasis. Methods: Exosomes isolated from EO771 TNBC cell line (EO771/exo) and HC11 normal breast epithelial cell line (HC11/exo) were characterized. Uptake of exosomes by macrophages was visualized by confocal microscopy. Mass spectrometry was applied to identify the significantly highly expressed HMGB1 protein in EO771/exo, and ligands of HMGB1 on lung macrophages were detected by co-immunoprecipitation. Functions of EO771/exo and HMGB1 in inducing PD-L1+-TAMs and generating a pre-metastatic immunosuppressive niche were confirmed by both in vitro and in vivo studies. Roles of EO771/exo and HMGB1 on glycolysis and lactate production were evaluated by metabolism assays. Associations between PD-L1+-TAMs and CD3+CD8+ T lymphocytes within TNBC axillary lymph nodes and lung metastasis specimens were analyzed by immunofluorescence and immunohistochemistry. Results: Confocal microscopy showed constant internalization and absorption of EO771/exo by macrophages. EO771/exo could polarize macrophages toward PD-L1+-TAMs and generate a pre-metastatic immunosuppressive niche, with respect to HC11/exo. Mass spectrometry, clinical samples, and bioinformatics analysis revealed that HMGB1 was highly expressed in EO771/exo and was involved in cellular communication and metabolism processes. Co-immunoprecipitation and 3-dimensional modeling indicated a strong binding of HMGB1 with Tim-3 ligand on lung macrophages. Macrophages treated with EO771/exo displayed an enhanced glycolysis and lactate production. Flow cytometry demonstrated that green fluorescent protein (GFP)-EO771 tumor-bearing mice had more lung micro-metastases than brain and liver. Immunofluorescence and laser confocal microscopy confirmed that EO771/exo, with respect to HC11/exo, were able to increase lung micro-metastases and escalate PD-L1 expression on lung macrophages. Moreover, elevated PD-L1+-TAMs and reduced CD3+CD8+ T lymphocytes were found in TNBC positive axillary lymph nodes and lung metastasis specimens than TNBC negative axillary lymph nodes. Conclusions: Exosomal HMGB1 from TNBC could target Tim-3 ligand on lung macrophages and accelerate glycolysis and lactate production to induce PD-L1+-TAMs, thereby suppressing CD3+CD8+ T lymphocytes immunity and generating a pre-metastasis immune-suppressive niche. Our research would uncover a novel mechanism of lung-tropic metastasis and provide a new therapeutic approach for TNBC treatment. Keywords: breast cancer; lung metastasis; exosomes; tumor-associated macrophages; metabolic reprogramming Citation Format: Wei-Xian Chen, Jia-Hao Wu, Tian-Cheng Cheng, Sujin Yang. Exosomal HMGB1 induce PD-L1+-tumor associated macrophages through glycometabolic reprogramming to promote lung-tropic metastasis of triple negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-06-11.

Unacylated Ghrelin Protects Against Muscle Wasting, Mitochondrial Dysfunction, and Neuromuscular Junction Disruption in Tumor Bearing Mice

Background: Cancer cachexia is a complex metabolic syndrome that severely impacts mobility, treatment strategies, and life quality of the patients. However, no treatments are available to mitigate the debilitating consequences of cancer cachexia. Ghrelin is a hormone released from the stomach that increases appetite upon acylation by binding to GHSR1a receptors in the brain. Because of its orexigenic effects and increases in body weight, ghrelin GHSR1a receptor analogs were tested as clinical trials to mitigate muscle wasting and loss of strength in cancer patients in several countries. The analogs (i.e., Anamorelin) increased body weight of the non-small lung cancer patients in European countries and Japan, but failed to increase strength, which is the critical component in cancer cachexia. Mainly due to the lack of improvement in strength and patients’ quality of life, Anamorelin was not approved for clinical use in Europe. While the lack of increases in strength is, at least in part, associated with the adipogenic effects of acylated ghrelin, unacylated ghrelin directly improves satellite cell renewal, muscle growth, and mitochondrial function independent of GHSR1a receptor activation and increases in appetite. Unacylated ghrelin is the predominant population in circulation comprising proximately 90-95% of total ghrelin in humans and rodents. Hypothesis: Unacylated ghrelin projects against muscle wasting, loss of strength, and metabolic dysfunction of tumor bearing mice. Methods and Results: Supplementing the cancer cell conditioned medium (CM) with unacylated ghrelin preserved myotube size. Unacylated ghrelin also diminished the cancer cell CM-induced upregulation of MuRF-1, muscle specific ubiquitin ligase. Interestingly, unacylated ghrelin normalized transcriptional upregulation of denervation markers (i.e., sarcolipin, Runx1, GADD45a) in myotubes treated with CM. To further investigate the therapeutic effects of unacylated ghrelin in vivo, we inoculated lung cancer cell line (LL2, ATCC®) in the flank of 4 months old male mice. Two weeks after the injection prior to development of cachexia, mice were provided with unacylated ghrelin containing drinking water or untreated water as control. Tumor bearing (TB) mice treated with unacylated ghrelin showed 30-40% increases in gastrocnemius and quadriceps weights, while soleus muscle mass was unchanged. In addition to the muscle quantity, unacylated ghrelin increased force generating capacity was also improved by unacylated ghrelin. Notably, unacylated ghrelin increased mitochondrial oxygen consumption rate and protected against neuromuscular junction disruption and denervation in TB control mice. Conclusion: Unacylated ghrelin protects against cancer cachexia by targeting multiple risk factors in skeletal muscle wasting, including protein balance, metabolic alterations, and neuromuscular junction disruption. Our findings show protective effects of unacylated ghrelin against cachexia and neuromuscular impairment in tumor bearing mice, indicating its therapeutic potential in cancer patients. NIA R00 064143 to Ahn B. Pilot funds from the Center for Redox Biology in Medicine, and Cancer Genetics and Metabolism program at the Wake Forest Baptist Comprehensive Cancer Center. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Expression of the TGF-β Target Gene Mss51 Is Induced in Muscle From Tumor-Bearing Mice and Attenuated by Treatment With the AMPK Activator AICAR

Cachexia is a severe complication of cancer characterized by skeletal muscle atrophy and loss of function and is associated with a poor prognosis. AMPK is a cellular energy sensor that is upregulated in cancer cachexia. AMPK activation is well-established in promoting catabolic cell signaling in skeletal muscle, partly through the induction of muscle-specific ubiquitin ligases MAFBx and MuRF-1 expression. Despite AMPK’s acute catabolic effects, chronic treatment with the AMPK-activating drug 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) has been reported to preserve muscle function and metabolism in various muscle-wasting conditions, including cancer cachexia. Elevated TGF-β during cancer cachexia is known to disrupt mitochondrial homeostasis and lead to mitochondrial dysfunction. Mss51 is a skeletal muscle-specific mitochondrial gene that is upregulated through TGF-β signaling. Although the role of Mss51 in skeletal muscle cancer cachexia has not been studied as of yet, its expression is associated with mitochondrial and metabolic impairment. Therefore, we hypothesized that Mss-51 expression would be elevated in cachexic skeletal muscle and attenuated by chronic AMPK activation. To test this, we inoculated mice (n=4) with either Lewis Lung Carcinoma (LLC) cells or PBS. Mice were injected daily with 350 mg/kg AICAR or an equivalent volume of saline beginning seven days after inoculation. Twenty-four days after inoculation, the mice were euthanized, and tissues were harvested and weighed. RNA was isolated from the gastrocnemius-plantaris-soleus complex and analyzed by RT-PCR for Mss51, MuRF-1, and MAFBx expression. Protein expression for hexokinase II (HKII) was measured by western blotting. AICAR treatment did not attenuate muscle atrophy in tumor-bearing mice. Compared to the control, Mss-51 was elevated in LLC mice treated with saline and decreased in LLC mice treated with AICAR, consistent with the known effects of AMPK in blocking TGF-β signaling. Similarly, MuRF-1 and MAFBx tended to be elevated in LLC-saline. Still, this increase was attenuated in LLC-AICAR mice, though we were likely underpowered to detect significance in this measure. In conclusion, based on previous work, the rise in Mss51 in tumor-bearing mice may contribute to mitochondrial and metabolic dysfunction in cancer cachexia. The prevention of this increase by AICAR suggests that AMPK may benefit dysfunctional metabolism in the cachexic muscle through inhibition of Mss51. Funding for these experiments was from a BYU Widtsoe Grant. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Continued use of alcohol accelerates cancer cachexia and impairs ability to induce 4E-BP1 but not p70S6K phosphorylation following muscle contraction

A primary co-morbidity of cancer is cancer cachexia characterized by the wasting of adipose tissue and skeletal muscle. Up to 80% of cancer patients may suffer from this wasting condition leading to reduced quality of life, poor treatment effectiveness and increased morbidity and mortality. The risk of cancer cachexia is enhanced in those with poor lifestyle habits including the habitual consumption of alcohol both prior to a cancer diagnosis and when consumption is continued during disease progression. The use of muscle contraction to improve outcomes in cachexia has been explored in alcohol naïve models with some success, however alcohol at high doses induces anabolic resistance preventing protein synthesis in response to an anabolic stimuli like muscle contraction. Therefore, muscle contraction may not be a suffcient stimuli to overcome cachexia induced catabolism when alcohol has been or is currently being consumed. The purpose of this work was to determine if alcohol induces anabolic resistance in tumor bearing mice or whether it can be used as a therapeutic intervention. It was hypothesized that alcohol would inhibit the stimulation of markers of the anabolic mTORC1 pathway in tumor bearing mice. METHODS: Female BALB/c mice, 12 weeks of age, were randomized into 4 groups (n = 12-14/group): control diet (CON), control diet-cancer (Cancer), Prior EtOH diet-cancer (PE-Cancer), and EtOH diet-cancer (E-cancer). All mice received the isocaloric Lieber DeCarli liquid diet with EtOH mice consuming 20% kcal/daily EtOH for 6 weeks. After 6 weeks, PE-cancer switched to the non-alcohol diet and all cancer groups had C26 carcinoma cells implanted. A single bout of maximal stimulated muscle contraction (10sets of 6 repetitions at 100Hz) was performed ~2 weeks later and muscles were collected 4 hours post contraction. RESULTS: E-cancer lost ~10% body mass, while no other cancer groups exhibited a significant loss in body mass indicating early-stage cachexia. Spleen mass was elevated in all cancer groups relative to control, while quadricep and gastrocnemius muscle weights were reduced in PE-cancer and E-cancer compared to CON. Muscle contraction significantly increased the phosphorylation of S6K1 Thr389 in all of groups except for PE-cancer where a strong trend was still detected (p=0.06). In contrast, phosphorylation of 4EBP1 Ser65 was only increased by muscle contraction in CON, as cancer prevented its stimulation. Therefore, despite accelerating body weight loss, continued alcohol consumption or prior alcohol consumption did not completely impair the anabolic response to maximally stimulated muscle contraction during the early stages of cancer cachexia. However, measurement of rates of degradation and synthesis over time are required to make further conclusions as to its therapeutic potential. Florida Department of Health Grant # 9BC03. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Epigallocatechin gallate protects against fat and muscle atrophy in B16BL6 melanoma-bearing mice on a high-fat diet

AimsEpidemiological evidence indicates that there is a substantial association between body mass index (BMI) and at least ten forms of cancer, including melanoma, and BMI imbalance contributes to the poor survival rate of cancer patients before and after therapy. Nevertheless, few pharmacological studies on models of obesity and cancer have been reported. In this study, we administered epigallocatechin gallate (EGCG) to B16BL6 tumor-bearing mice that received a high-fat diet (HFD) to examine its impact. MethodsB16BL6 tumor-bearing mice were fed a HFD. Body weight and food intake were documented every week. We conducted a Western blot analysis to examine the protein levels in the tumor, gastrocnemius (GAS), and tibialis anterior (TA) muscles, as well as the inguinal and epididymal white adipose tissues (iWAT and eWAT). Key findingsEGCG has been shown to have anti-cancer effects equivalent to those of cisplatin, a chemotherapy drug. Furthermore, EGCG protected against the loss of epidydimal white adipose tissue by regulating protein levels of lipolysis factors of adipose triglyceride lipase and hormone-sensitive lipase as well as WAT browning factors of uncoupling protein 1, as opposed to cisplatin. EGCG was shown to reduce the protein levels of muscular atrophy factors of muscle RING-finger protein-1, whereas cisplatin did not contribute to rescuing the atrophy of TA and GAS muscles. ConclusionTaken together, our findings indicate that EGCG has a preventive effect against cachexia symptoms and has anti-cancer effects similar to those of cisplatin in tumor-bearing mice fed a high-fat diet.

Combining toll-like receptor agonists with immune checkpoint blockade affects antitumor vaccine efficacy

BackgroundT cell checkpoint receptors are expressed when T cells are activated, and modulation of the expression or signaling of these receptors can alter the function of T cells and their...

Photothermal therapy of xenografted tumor by carbon nanoparticles-Fe(II) complex

Due to the unique structure, carbon nanomaterials could convert near-infrared (NIR) light into heat efficiently in tumor ablation using photothermal therapy (PTT). However, none of them has been applied in clinical treatment, because they have not been approved for clinical evaluations and the precise temperature control facility is scarce. In this study, we designed a temperature-responsive controller for PTT and used carbon nanoparticles-Fe(II) complex (CNSI-Fe) as photothermal conversion agent (PTA) for PTT of tumor in vitro and in vivo. CNSI-Fe was an innovative drug under the evaluations in clinical trials. CNSI-Fe showed excellent photothermal conversion ability in water to increase the water temperature by 40 °C within 5min under irradiation of 808nm laser at 0.5W/cm2. The temperature was precisely controlled at 52 °C for both in vitro and in vivo tumor inhibition. CNSI-Fe with NIR irradiation showed higher tumor cell inhibition than CNSI. In tumor bearing mice, CNSI-Fe with NIR irradiation achieved an inhibition rate of 84.7% and 71.4% of them were completely cured. Mechanistically, CNSI-Fe under NIR irradiation induced the radical generation, oxidative damage and ferroptosis to kill tumor. In addition, CNSI-Fe showed good biosafety during PTT according to hematological, serum biological and histopathological examinations. These results indicated that the combination of chemotherapy and PTT provided higher antitumor efficiency using CNSI-Fe as PTA.

PGE2 induced miR365/IL-6/STAT3 signaling mediates dendritic cell dysfunction in cancer

AimTo understand the mechanism of prostaglandin E2 (PGE2)-mediated immunosuppression in dendritic cells (DCs). Main methodsIn vivo experiments were conducted on 4T1 tumor bearing mice (TBM). In vitro experiments were performed in bone marrow-derived DCs (BMDCs), or spleen cells. Cytokines were monitored by ELISA/ELIspot. Gene expression was monitored by RT-PCR/flow cytometry. Key findingsIn silico, in vitro, and in vivo experiments in 4T1 TBM revealed that PGE2 induced IL-6/pSTAT3 signaling through EP4 receptors in DCs, resulting in their dysfunction. These effects were reversed by EP4 antibody neutralization, EP4 antagonist, and STAT3 inhibitory peptides. PGE2 induced IL-6 could be regulated by miR-365, as its mimic inhibited PGE2 induced IL-6 and the inhibitor increased lL-6 levels in DC. Bio-informatic analysis in human mammary cancers also revealed a strong compared co-relation between PGE2 and IL-6 (Correlation AnalyzeR) (R = 0.94). Mice bearing PTGS-2 KD 4T1 tumors had decreased tumor burden, PGE2, EP4, IL-6, and pSTAT3 signaling, along with improved DCs and T cell functions. Treatment of mice with a cyclooxygenase-2 (COX-2) inhibitor or EP4 antagonist decreased tumor burden, and this effect of EP4 antagonist was abrogated upon in vivo depletion of CD11c cells, indicating the crucial role of PGE2 signaling in DCs in tumor progression. SignificanceIn summary, our data highlights the importance of dendritic cells in mediating PGE2-mediated immunosuppression and EP4 or STAT3 inhibitors or the use of miR365 mimics can restore immunogenicity in cancer.