Reduced Tumor Growth Rate Research Articles

Hsp70-containing extracellular vesicles are capable of activating of adaptive immunity in models of mouse melanoma and colon carcinoma

The release of Hsp70 chaperone from tumor cells is found to trigger the full-scale anti-cancer immune response. Such release and the proper immune reaction can be induced by the delivery of recombinant Hsp70 to a tumor and we sought to explore how the endogenous Hsp70 can be transported to extracellular space leading to the burst of anti-cancer activity. Hsp70 transport mechanisms were studied by analyzing its intracellular tracks with Rab proteins as well as by using specific inhibitors of membrane domains. To study Hsp70 forms released from cells we employed the assay consisting of two affinity chromatography methods. Hsp70 content in culture medium and extracellular vesicles (EVs) was measured with the aid of ELISA. The properties and composition of EVs were assessed using nanoparticle tracking analysis and immunoblotting. The activity of immune cells was studied using an assay of cytotoxic lymphocytes, and for in vivo studies we employed methods of affinity separation of lymphocyte fractions. Analyzing B16 melanoma cells treated with recombinant Hsp70 we found that the chaperone triggered extracellular transport of its endogenous analog in soluble and enclosed in EVs forms; both species efficiently penetrated adjacent cells and this secondary transport was corroborated with the strong increase of Natural Killer (NK) cell toxicity towards melanoma. When B16 and CT-26 colon cancer cells before their injection in animals were treated with Hsp70-enriched EVs, a powerful anti-cancer effect was observed as shown by a two-fold reduction in tumor growth rate and elevation of life span. We found that the immunomodulatory effect was due to the enhancement of the CD8-positive response and anti-tumor cytokine accumulation; supporting this there was no delay in CT-26 tumor growth when Hsp70-enriched EVs were grafted in nude mice. Importantly, pre-treatment of B16 cells with Hsp70-bearing EVs resulted in a decline of arginase-1-positive macrophages, showing no generation of tumor-associated macrophages. In conclusion, Hsp70-containing EVs generated by specifically treated cancer cells give a full-scale and effective pattern of anti-tumor immune responses.

Abstract 2790: Temporal immune changes of murine breast and melanoma tumor microenvironments following pulsed focused ultrasound

Abstract Various cellular and biological immunotherapies have revolutionized the treatment of human malignancies by augmenting endogenous immune responses within the tumor microenvironment (TME). Although non-ablative pulsed-FUS (pFUS) has received less attention, it has been successfully used as a noninvasive treatment of solid malignancies by stimulating a pro-inflammatory immune response within the TME. Such an approach could be useful to treat peripheral micro-metastases in normal-appearing tissues. In the present study, we interrogated the immune-modulatory changes that occur within the TME following pFUS exposure. Murine B16 melanoma and 4T1 mammary carcinoma cells were subcutaneously implanted into both mice flanks (n=10 tumors/group/time point). The natural histories cytokine, chemokine, trophic factor (CCTF), and cell adhesion molecule (CAM) expression was evaluated within the TME of untreated flank tumors over 11 days. Ultrasound-guided pFUS at 1 MHz and peak negative pressures of 6 MPa was administered to tumors after reaching ~5 mm in diameter. Temporal immune-related changes in tumors were assessed following sonication using using proteomic, transcriptomic and histological analyses. Natural history growth characteristics showed a progressive increase in size for both tumors, and proteomic analysis revealed a shift toward an immunosuppressive TME. An anti-tumor TME was detected following pFUS treatment with increased expression of various pro-inflammatory CCTF and CAM as well as reduced tumor growth rates. Transcriptomic analyses following sonication identified inverted gene expression patterns in the two tumor types. Functional analysis of B16 tumors revealed increased intracellular signaling pathways associated with immune response regulations, while 4T1 tumors demonstrated reduced expression of proliferation genes. This study provides a macroscopic overview on the temporal dynamics of the TME and underline the profound magnitude of tumoral heterogeneity between tumor types as well as through progressive stages of similar primary tumors. Nevertheless, the induced immune-modulation changes suggest that pFUS may alter the expression of pro-inflammatory CCTF, CAM, towards an anti-tumoral TME, and supports the potential use of pFUS as a neoadjuvant treatment approach. Citation Format: Gadi Cohen, Parwathy Chandran, Rebecca M. Lorsung, Lauren E. Tomlinson, Robert B. Rosenblatt, Scott R. Burks, Joseph A. Frank. Temporal immune changes of murine breast and melanoma tumor microenvironments following pulsed focused ultrasound [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2790.

A phase II, open-label, randomized trial of durvalumab (D) with olaparib (O) or cediranib (C) in patients (pts) with leiomyosarcoma (LMS).

11522 Background: The use of immune checkpoint blockade (ICB) in non-inflamed (cold) tumors is associated with limited clinical efficacy. Combination of ICB with certain molecularly targeted agents (MTA) is hypothesized to increase tumor immunogenicity by recruiting tumor infiltrating lymphocytes in cold tumors, such as LMS. Here, we present the results of LMS cohort treated on the DAPPER study (NCT03851614). Methods: LMS pts with ECOG 0-1 were randomized to either D+O (arm A), or D+C (arm B). In a 28-day cycle, D 1500mg i.v. q4w with either O 300mg bid po qd or C 20mg po qd 5d/week were administered. Overall response rates (ORR) were determined using RECISTv1.1. Evaluation of tumor kinetics (TK) was performed by calculating tumor growth rate (TGR) of target lesions on CT images at baseline and on-treatment, adjusted to account for the time difference between scans. TGR is expressed as % tumor growth/week (Ferte C et al. CCR, 2014). Additionally, paired FFPE samples (from baseline and on-treatment biopsies) were assessed using multispectral fluorescent immunohistochemistry (IHC) panel: CD3, CD8, CD20, CD68, FOXP3 and cytokeratin. Tumor areas were identified by a pathologist and immune cells were quantified using InForm image analysis software. Results: 25 metastatic LMS pts were randomized to arm A (n = 11) or B (n = 14) over 21 months. Median age was 53 years, 96% were females and 60% of pts had ≥3 lines of therapy. In 23 evaluable pts, no responses were seen, 7 pts had stable disease (SD) while 16 has progressive disease (PD). TK analysis was evaluable for 18 pts (arm A = 8, B = 10). 5/8 pts (62.5%) in arm A and 6/10 pts (60%) in arm B showed decreased TK (defined as TGRbaseline > TGRon-treatment). In 4/5 (80%) pts who had deceleration of TK in arm A, SD was maintained for ≥6 months. The reduction in TGR on treatment, compared to baseline was significant in arm A but not in arm B (measured as median % tumor growth/week of 0.5 vs 5.1, 95% CI 0.2-4.3, p = 0.035 in arm A; and 1.3 vs 2.9, 95% CI 0.2-2.7, p = 0.088 in arm B). The median PFS of arm A and B were 9 (95% CI 3-12.8) and 4 (95% CI 2.2-4.6) months respectively. There were no statistically significant differences in tumor-infiltrating immune cells when comparing baseline and on-treatment biopsies from arm A or B. In arm A, one pt with SD > 6 months had a 2.5-fold increase in CD8 (CD3+CD8+) T cells and a 7.6-fold increase in macrophages (CD68+). Conclusions: D+O or D+C resulted in stable disease in 30% of pts, mostly on arm A (D+O). TK analysis may identify pts with prolonged SD on treatment. Although a cold-to-hot immunophenotype change was not generally seen, changes in tumor infiltrating immune cell subsets were observed in one patient with prolonged stable disease. These findings support further molecular and immunophenotype characterization in LMS patients treated with D+O or D+C. Clinical trial information: NCT03851614.

A recombinant Newcastle disease virus expressing MMP8 promotes oncolytic efficacy

Oncolytic virus is an emerging anti-cancer strategy. However, extracellular matrix (ECM), as a physical barrier, limits virus spread within the tumor. To overcome the obstacle, we constructed a recombinant Newcastle disease virus (NDV) expressing matrix metalloproteinase (MMP8) (NDV-MMP8) using with reverse genetic technology. In vitro, NDV-MMP8 was identified and verified by WB and ELISA. Cell viability was detected by CCK-8 assay. In vivo, we established two liver cancer xenograft models. NDV-MMP8 was injected into the tumor to observe the tumor volume and survival of mice. The changes in extracellular matrix were observed by Masson's trichrome staining. Virus expression in tumor tissues was detected by immunofluorescence assay. The virus titer in tumor tissues was detected by TCID50. Histopathological changes were detected by hematoxylin and eosin (HE) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Intratumoral administration of NDV-MMP8 can effectively degrade ECM, promote the spread of the virus within the tumor, and reduce tumor growth rate. Therefore, the method of increasing intratumoral virus accumulation by degradation of the ECM to enhance the oncolytic effect has great potential for clinical application.

Veliparib Is an Effective Radiosensitizing Agent in a Preclinical Model of Medulloblastoma.

Medulloblastoma is the most common malignant childhood brain tumor, and 5-year overall survival rates are as low as 40% depending on molecular subtype, with new therapies critically important. As radiotherapy and chemotherapy act through the induction of DNA damage, the sensitization of cancer cells through the inhibition of DNA damage repair pathways is a potential therapeutic strategy. The poly-(ADP-ribose) polymerase (PARP) inhibitor veliparib was assessed for its ability to augment the cellular response to radiation-induced DNA damage in human medulloblastoma cells. DNA repair following irradiation was assessed using the alkaline comet assay, with veliparib inhibiting the rate of DNA repair. Veliparib treatment also increased the number of γH2AX foci in cells treated with radiation, and analysis of downstream pathways indicated persistent activation of the DNA damage response pathway. Clonogenicity assays demonstrated that veliparib effectively inhibited the colony-forming capacity of medulloblastoma cells, both as a single agent and in combination with irradiation. These data were then validated in vivo using an orthotopic implant model of medulloblastoma. Mice harboring intracranial D425 medulloblastoma xenografts were treated with vehicle, veliparib, 18 Gy multifractionated craniospinal irradiation (CSI), or veliparib combined with 18 Gy CSI. Animals treated with combination therapy exhibited reduced tumor growth rates concomitant with increased intra-tumoral apoptosis observed by immunohistochemistry. Kaplan–Meier analyses revealed a statistically significant increase in survival with combination therapy compared to CSI alone. In summary, PARP inhibition enhanced radiation-induced cytotoxicity of medulloblastoma cells; thus, veliparib or other brain-penetrant PARP inhibitors are potential radiosensitizing agents for the treatment of medulloblastoma.

Chronic Adrenergic Stress Contributes to Metabolic Dysfunction and an Exhausted Phenotype in T Cells in the Tumor Microenvironment.

Metabolic dysfunction and exhaustion in tumor-infiltrating T cells have been linked to ineffectual antitumor immunity and the failure of immune checkpoint inhibitor therapy. We report here that chronic stress plays a previously unrecognized role in regulating the state of T cells in the tumor microenvironment (TME). Using two mouse tumor models, we found that blocking chronic adrenergic stress signaling using the pan β-blocker propranolol or by using mice lacking the β2-adrenergic receptor (β2-AR) results in reduced tumor growth rates with significantly fewer tumor-infiltrating T cells that express markers of exhaustion, with a concomitant increase in progenitor exhausted T cells. We also report that blocking β-AR signaling in mice increases glycolysis and oxidative phosphorylation in tumor-infiltrating lymphocytes (TIL), which associated with increased expression of the costimulatory molecule CD28 and increased antitumor effector functions, including increased cytokine production. Using T cells from Nur77-GFP reporter mice to monitor T-cell activation, we observed that stress-induced β-AR signaling suppresses T-cell receptor (TCR) signaling. Together, these data suggest that chronic stress-induced adrenergic receptor signaling serves as a "checkpoint" of immune responses and contributes to immunosuppression in the TME by promoting T-cell metabolic dysfunction and exhaustion. These results also support the possibility that chronic stress, which unfortunately is increased in many patients with cancer following their diagnoses, could be exerting a major negative influence on the outcome of therapies that depend upon the status of TILs and support the use of strategies to reduce stress or β-AR signaling in combination with immunotherapy.

The MEK1/2 Pathway as a Therapeutic Target in High-Grade Serous Ovarian Carcinoma.

Simple SummaryHigh-grade serous ovarian carcinoma (HGSOC) has poor prognosis for patients due to its high rate of recurrence and acquired resistance to therapy. MEK1/2-ERK1/2 signaling pathway that controls cell proliferation and survival is active in the majority of HGSOC cases, but its functional impact is unclear. We suggest that inhibition of MEK1/2 with specific inhibitor trametinib may exert anti-cancer effects upon HGSOC cells. Here we demonstrate that trametinib treatment of HGSOC cells indeed prominently inhibits cell proliferation and tumor growth, and that cisplatin-resistant cells displaying high MEK1/2 activity are particularly sensitive to trametinib. However, we also discovered that trametinib treatment of HGSOC cells has no cytotoxic effects and promotes cancer stem-like characteristics. We therefore suggest to use MEK1/2 inhibitors with other treatment strategies targeting cancer stem-like cells, like aldehyde dehydrogenase 1 inhibition that might show together strong synergy.High-grade serous ovarian carcinoma (HGSOC) is the deadliest of gynecological cancers due to its high recurrence rate and acquired chemoresistance. RAS/MEK/ERK pathway activation is linked to cell proliferation and therapeutic resistance, but the role of MEK1/2-ERK1/2 pathway in HGSOC is poorly investigated. We evaluated MEK1/2 pathway activity in clinical HGSOC samples and ovarian cancer cell lines using immunohistochemistry, immunoblotting, and RT-qPCR. HGSOC cell lines were used to assess immediate and lasting effects of MEK1/2 inhibition with trametinib in vitro. Trametinib effect on tumor growth in vivo was investigated using mouse xenografts. MEK1/2 pathway is hyperactivated in HGSOC and is further stimulated by cisplatin treatment. Trametinib treatment causes cell cycle arrest in G1/0-phase and reduces tumor growth rate in vivo but does not induce cell death or reduce fraction of CD133+ stem-like cells, while increasing expression of stemness-associated genes instead. Transient trametinib treatment causes long-term increase in a subpopulation of cells with high aldehyde dehydrogenase (ALDH)1 activity that can survive and grow in non-adherent conditions. We conclude that MEK1/2 inhibition may be a promising approach to suppress ovarian cancer growth as a maintenance therapy. Promotion of stem-like properties upon MEK1/2 inhibition suggests a possible mechanism of resistance, so a combination with CSC-targeting drugs should be considered.

Abstract PO018: Restoring the tumor-suppressed immune response during chemotherapy by targeting Mer:PTP1b interactions

Abstract The development of adaptive immune checkpoint inhibitors, which prevent tumor-mediated inactivation of lymphocytes, has drastically improved outcomes for advanced cancer patients. However, only 20-30% of patients benefit from these treatments and those that do often present moderate toxicity and/or later relapse. After discovering a novel innate immune checkpoint, we have identified a therapeutic strategy to restore the tumor-suppressed immune response during chemotherapy. Here, we show that multiple tumor types (including melanoma, lung, breast and pancreatic cancers) co-cultured with murine primary macrophages suppress the expression of the essential Toll-like Receptor (TLR) signal-transducing adapter protein MyD88 by 57-81% +/- 2-5% (mean +/- SEM). In the absence of MyD88, macrophages are unable to respond to TLR stimulating Damage Associated Molecular Patterns (DAMPs) released by dying tumor cells after chemotherapy, leading to as much as 97% +/- 6% reduction in pro-inflammatory gene expression in our co-culture models. Tumors exert this suppression by activating the macrophage Mer receptor, inducing recruitment of a phosphatase, known as PTP1b, and Stat1 to Mer. Increasing association between PTP1b and Stat1 leads to reduced Stat1 phosphorylation and translocation to the nucleus as well as a downstream reduction in MyD88 expression. Treatment with a pharmacological inhibitor of PTP1b (BVT948) blocks the association of Mer, PTP1b and Stat1, restoring MyD88 expression and responsiveness to chemotherapy-released DAMPs. In chemotherapy-resistant murine syngeneic melanoma models, including B16F10 and GEMM6 (Braf V600E, Pten-/-), we show that combination therapy with BVT948 and Cisplatin or Vemurafenib, respectively, leads to a 47% reduction in tumor growth rates. Treating chemo- and immuno-therapy resistant Lewis Lung Cancer (LLC) tumor-bearing mice with combination BVT948/Cisplatin therapy leads to a similar reduction in tumor growth that is magnified with the addition of adaptive immune checkpoint blockade. In summary, we have described a novel mechanism of cancer-mediated immune suppression that dramatically reduces the immune response during chemotherapy. PTP1b inhibition could represent a novel therapeutic strategy to improve the effectiveness of conventional chemotherapy in patients with advanced malignancies. Citation Format: Nestor Prieto-Dominguez, Eric S. Ubil. Restoring the tumor-suppressed immune response during chemotherapy by targeting Mer:PTP1b interactions [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO018.

Anti-epithelial cell adhesion molecule RNA aptamer-conjugated liposomal doxorubicin as an efficient targeted therapy in mice bearing colon carcinoma tumor model.

To overcome the lack of selectivity and nonspecific biodistribution of drugs in the body, targeted delivery of chemotherapeutic agents with aptamers is a very effective method. In this strategy, aptamers could be specifically identified and attach to targeted molecules on the cancerous cells and deliver the chemotherapeutic agents to desired tissue with minimal or no damage to the normal cells. In this study, we designed anti-epithelial cell adhesion molecule (EpCAM) RNA aptamer conjugated PEGylated liposomal doxorubicin (ER-lip) to investigate its in vitro and in vivo anticancer abilities. Data showed that EpCAM aptamer was able to enhance cell uptake and cytotoxic effects of Dox in C26 cell line. The biodistribution study indicated that ER-lip enhanced the tumor accumulation of Dox compared to Caelyx. Also, double staining of isolated tumor cells with anti-CD44-PE-cy5 and anti-EpCAM Cy-7 antibodies indicated that tumor cells expressed a high level of EpCAM+ CD44+ cells (p≤.001) compared to cultured C26 cell line. in vivo results showed that ER-lip promoted survival and reduced tumor growth rate in animal model compared to Caelyx. In conclusion, these results suggested that the ER-lip could be served as promising formulation for the treatment of cancers with the high expression of EpCAM.

HOXC-AS2 mediates the proliferation, apoptosis, and migration of non-small cell lung cancer by combining with HOXC13 gene

ABSTRACT Non-small cell lung cancer (NSCLC) is the highest incidence and mortality of malignant tumors worldwide and has become a global public health problem. Long non-coding RNAs (LncRNAs) are expected to participate in the progression of NSCLC. This study aims to explore the effects and underlying mechanisms of LncRNA HOXC-AS2 on NSCLC cell proliferation, apoptosis, and migration. The Cell Counting Kit-8 (CCK-8) and clone formation assay were used to measure the A549 and HCC827 cell proliferation. The cell apoptosis and migration was respectively analyzed by flow cytometry and transwell assay. RNA immunoprecipitation (RIP) was used to detect the interaction between HOXC-AS2 and HOXC13. The expression of β-catenin, α-SMA, MMP-1, MMP-2 expression, E-cadherin, and Ki-67 expression were determined by Western blot or immunohistochemistry (IHC) assay. We found that HOXC-AS2 was significantly up-regulated in NSCLC tissues. Knockdown of HOXC-AS2 expression resulted in significant decreases in NSCLC cell proliferation, migration, and epithelial-mesenchymal transition (EMT) process marker proteins, simultaneously activated A549 and HCC827 cell apoptosis. RIP assay suggested that HOXC13 was a functional target for HOXC-AS2. And HOXC-AS2 and HOXC13 could positively regulate each other. Compared with the normal tissues, the mRNA level of HOXC13 was increased in NSCLC tissues. HOXC13 silencing counteracted increases of A549 and HCC827 cell proliferation and migration, as well as a decrease of cell apoptosis induced by HOXC-AS2 overexpression. Moreover, HOXC-AS2 silencing reduced tumor growth rate and Ki-67 expression in vivo. Taken together, HOXC-AS2 knockdown inhibited NSCLC cell proliferation and migration, as well as stimulated NSCLC cell apoptosis through regulation of HOXC13 expression.

SPION and doxorubicin-loaded polymeric nanocarriers for glioblastoma theranostics.

Glioma is a type of cancer with a very poor prognosis with a survival of around 15months in the case of glioblastoma multiforme (GBM). In order to advance in personalized medicine, we developed polymeric nanoparticles (PNP) loaded with both SPION (superparamagnetic iron oxide nanoparticles) and doxorubicin (DOX). The former being used for its potential to accumulate the PNP in the tumor under a strong magnetic field and the later for its therapeutic potential. The emulsion solvent and evaporation method was selected to develop monodisperse PNP with high loading efficiency in both SPION and DOX. Once injected in mice, a significant accumulation of the PNP was observed within the tumoral tissue under static magnetic field as observed by MRI leading to a reduction of tumor growth rate.

Knockdown of Annexin-A1 Inhibits Growth, Migration and Invasion of Glioma Cells by Suppressing the PI3K/Akt Signaling Pathway.

ANXA1, which can bind phospholipid in a calcium dependent manner, is reported to play a pivotal role in tumor progression. However, the role and mechanism of ANXA1 involved in the occurrence and development of malignant glioma are still not well studied. Therefore, we explored the effects of ANXA1 on normal astrocytes and glioma cell proliferation, apoptosis, migration and invasion and the underlying mechanisms. We found that ANXA1 was markedly up-regulated in glioma cell lines and glioma tissues. Down-regulation of ANXA1 inhibited normal astrocytes and glioma cell proliferation and induced the cell apoptosis, which suggested that the consequences of loss of Annexin 1 are not specific to the tumor cells. Furthermore, the siRNA-ANXA1 treatment significantly reduced tumor growth rate and tumor weight. Moreover, decreasing ANXA1 expression caused G2/M phase arrest by repressing expression levels of cdc25C, cdc2 and cyclin B1. Interestingly, ANXA1 did not affect the expressions of β-catenin, GSK-3β and NF-κB, the key signaling molecules associated with cancer progression. However, siRNA-ANXA1 was found to negatively regulate phosphorylation of AKT and the expression and activity of MMP2/-9. Finally, the decrease of cell proliferation and invasiveness induced by ANXA1 down-regulation was partially reversed by combined treatment with AKT agonist insulin-like growth factor-1 (IGF-1). Meanwhile, the inhibition of glioma cell proliferation and invasiveness induced by ANXA1 down-regulation was further enhanced by combined treatment with AKT inhibitor LY294002. In summary, these findings demonstrate that ANXA1 regulates proliferation, migration and invasion of glioma cells via PI3K/AKT signaling pathway.

Jervine exhibits anticancer effects on nasopharyngeal carcinoma through promoting autophagic apoptosis via the blockage of Hedgehog signaling

Nasopharyngeal carcinoma (NPC) is a malignant tumor originating from the superior mucosal epithelium of the nasopharynx. However, effective therapies for NPC are still required. Reducing Hedgehog signaling pathway has been shown to suppress tumor growth. In this study, we attempted to explore whether Jervine (JV), an inhibitor of Hedgehog signaling, had anti-cancer effects on NPC, and the underlying mechanisms. Our findings showed that JV treatments markedly reduced the proliferation of NPC cells in a dose- and time-dependent manner. Cell cycle arrest in G2/M phase was significantly enhanced by JV, along with evident DNA damage. Moreover, JV treatment effectively induced apoptosis in NPC cells through improving Caspase-3 activation. Furthermore, ROS production and mitochondrial impairments were detected in JV-incubated NPC cells with elevated releases of Cyto-c from mitochondria. JV also dramatically triggered autophagy through blocking AKT/mTOR and increasing AMPK signaling pathways. Intriguingly, we showed that JV-induced apoptosis was mainly via an autophagy-dependent manner. In addition, the expression levels of SHH, PTCH1, SMO and GLI1 were markedly suppressed in NPC cells, demonstrating the hindered Hedgehog signaling. Importantly, we found that JV-induced apoptosis and autophagy were closely associated with the blockage of Hedgehog signaling. Our in vivo studies confirmed the anti-cancer effects of JV on NPC through inducing autophagy, as evidenced by the markedly reduced tumor growth rate and weight without side effects and toxicity. Taken together, JV may be a promising and effective agent for human NPC treatment through repressing Hedgehog signaling pathway and inducing autophagic cell death.

POU4F1 confers trastuzumab resistance in HER2-positive breast cancer through regulating ERK1/2 signaling pathway

Over-expression of the human epidermal growth factor receptor-2 (HER2) is related to aggressive tumors and poor prognosis in breast cancer. Trastuzumab (TRA) resistance leads to tumor recurrence and metastasis, resulting in poor prognosis in HER2-positive breast cancer. POU Class 4 Homeobox 1 (POU4F1) is a member of the POU domain family transcription factors, and has a key role in regulating cancers. However, its effects on TRA-resistant HER2-positive breast cancer are still vague. In the present study, we found that POU4F1 expression was dramatically increased in clinical breast cancer specimens with TRA resistance. Higher POU4F1 was also detected in HER2-positive breast cancer cells with TRA resistance than that of the parental ones. Poor prognosis was detected in breast cancer patients with high POU4F1 expression. Under TRA treatment, POU4F1 knockdown significantly reduced the proliferative capacity of HER2-positive breast cancer cells with TRA resistance. POU4F1 silence also sensitized resistant HER-positive breast cancer cells to TRA treatment in vivo using a xenograft mouse model, along with the markedly reduced tumor growth rate and tumor weight. Moreover, we found that POU4F1 deletion greatly decreased the activation of mitogen-activated or extracellular signal-regulated protein kinase kinases 1 and 2 (MEK1/2) and extracellular-regulated kinase 1/2 (ERK1/2) signaling pathways in breast cancer cells with TRA resistance. Migration and invasion were also effectively hindered by POU4F1 knockdown in TRA-resistant HER2-positive breast cancer cells. Notably, we found that POU4F1 deletion-improved chemosensitivity of HER2-positive breast cancer cells with drug-resistance to TRA treatment was closely associated with the blockage of ERK1/2 signaling. Collectively, our findings reported a critical role of POU4F1 in regulating TRA resistance, and demonstrated the underlying molecular mechanisms in HER2-positive breast cancer. Thus, POU4F1 may be a promising prognostic and therapeutic target to develop effective treatment for overcoming TRA resistance.

Brucine PEGylated nanoemulsion: In vitro and in vivo evaluation

Cancer represents one of the most life threatening disease worldwide. Poly ethylene glycol nanoemulsion (PEG-NE), a new drug delivery system (DDS) is widely utilized for encapsulating water insoluble drugs and delivering it into cancerous tissue following its intravenous injection. Brucine (BRU) is an effective anti-carcinogenic agent toward different cancer cell lines; however, its low solubility represents a great obstacle in its formulation. The objective of this investigation was to develop and evaluate BRU-PEG-NE. Physicochemical evaluation including, viscosity, particle size and entrapment efficiency investigations were performed. Quantitative and qualitative estimation of total serum protein adsorbed onto the surface of BRU-NE were performed. in vitro release studies of BRU-NE with and without serum incubation were assayed. Formulations were tested for their cytotoxic activity using MTT assay. Finally, in-vivo anticancer effect was evaluated in tumor-inoculated mice. Homogenous NEs were obtained with particle size less than 140 nm and viscosity less than 3.3 cP for BRU-PEG-NE. Total serum protein adsorbed was less than 17.33 ± 0.76 μg/μmol total lipid for BRU-PEG-NE. in vitro drug release was less than 65 % over 24 h for PEG-NE. As well, PEG-NEs could achieve significant inhibition for the viability of cancer cells. Finally, significant reduction in tumor growth rate and mortality rate for BRU PEG-NE formulations were obtained. The obtained results suggested that BRU PEG NE could be considered as a potential mean for cancer therapy.

Brucea javanica Increases Survival and Enhances Gemcitabine Efficacy in a Patient-derived Orthotopic Xenograft (PDOX) Mouse Model of Pancreatic Cancer

Traditional Chinese medicine (TCM) Brucea javanica (BJO) has shown anti-proliferation efficacy on human carcinoma cells in vitro. The aim of the present study was to evaluate for the first time the efficacy of BJO combined with the first-line chemotherapeutic drug gemcitabine (GEM) on tumor growth-inhibition and survival in a pancreatic cancer patient-derived orthotopic xenograft (PDOX) mouse model. The pancreatic cancer tumor fragment originated from a patient at the Hefei First People's Hospital (Anhui, PR China). The surgical specimen was transplanted orthotopically in nude mice using surgical orthotopic implantation (SOI). All mice were randomized and assigned to 5 groups: G1: saline vehicle (0.1ml per mouse, oral, once per day); G2: GEM [100 mg/kg, intraperitoneal (i.p), twice per week]; G3: GEM+BJO [100 mg/kg GEM, i.p, twice per week+1g/kg BJO, oral, once per day (qd)]; G4: BJO (1g/kg, oral, qd). Group 5 and Group 6 were used to observe survival [G5: saline vehicle (0.1ml per mouse, oral, qd), G6: BJO (1g/kg, oral, qd)]. Body weight and tumor volume were measured twice per week. TUNEL staining was used to determine apoptosis. The combination of GEM + BJO resulted in a reduced tumor growth rate (p<0.05) and greater apoptosis (p<0.05) compared to the vehicle control and GEM monotherapy. In addition, the BJO-treated group showed a statistically significant increase in survival compared to the vehicle control (p<0.05). BJO is a promising non-toxic TCM to effectively treat pancreatic cancer, both as monotherapy and in combination with first-line GEM therapy.

Abstract 391: Lrg1 blockade modulates the tumor immune microenvironment and improves the efficacy of cancer therapeutics

Abstract Although the use of checkpoint inhibitors and adoptive T cell therapy have been reported in multiple cancers resulting in a plethora of clinical trials, the efficacy of these therapeutic strategies is often limited by the presence of impaired vasculature in the tumour mass. Indeed, in solid tumors, blood vessels are dysfunctional, leaky and poorly perfused, and thwart not only the efficacy of systemically derived drugs but also the extravasation of T cells, promoting an immunosuppressive microenvironment. Leucine-rich alpha-2-glycoprotein 1 (LRG1) is a secreted glycoprotein that is associated with pathogenic neovascularization, autoimmune diseases, chronic inflammation, and is significantly up-regulated in many solid cancers. We have observed that treatment with a LRG1 function-blocking antibody (15C4) significantly limits growth of solid tumors inducing vessel normalization and functionality. In the present study, we aim to investigate if LRG1 blockade enhances an immunostimulatory milieu, making tumors more responsive to current immunotherapies. Murine melanoma cells harbouring the internal influenza nucleoprotein antigen, NP68, were grafted subcutaneously before antigen-specific F5B6 CD8+ T cells were also transferred. Interestingly, the combination with 15C4 promoted a greater reduction in tumor growth rate. Moreover, not only was the number of donor CD8+ T cells significantly higher when delivered with 15C4, but also the total number of CD3+ T cells was increased following combination therapy, suggesting a more robust host-derived lymphocyte trafficking. As blocking LRG1 normalizes the tumor vasculature and enhances T cell extravasation, we then evaluated whether 15C4 could improve delivery and efficacy of an anti-PD1 checkpoint inhibitor. As monotherapies, both 15C4 and anti-PD1 antibodies induced a marked reduction in tumor volume but when the two were combined, a significant additive effect was also observed. Histological analysis revealed that the combination therapy was associated with a significantly higher number of activated CD8+ T cells, together with enhanced expression of Granzyme B. Current studies are investigating whether the observed boost in cytotoxic T cell density and functionality is a direct consequence of vessel normalization or whether LRG1 blockade is able to modulate the tumour immune microenvironment at different levels: for example, FACS analysis of tumour-infiltrating leukocytes revealed a reduction in the percentage of regulatory T cells after dual therapy, while histological analysis suggested an increase in the number of M1 macrophages. These data show that LRG1 blockade orchestrates a shift in the tumour microenvironment from being immune silent to immune active and, therefore, might represent a novel tool to enhance the efficacy of cancer therapies. Citation Format: Carlotta Camilli, Camilla Pilotti, Marie N. O'Connor, David Kallenberg, Markella Alatsatianos, Angharad H. Watson, Julia Ohme, Athina Dritsoula, Ann Ager, Stephen E. Moss, John Greenwood. Lrg1 blockade modulates the tumor immune microenvironment and improves the efficacy of cancer therapeutics [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 391.

Abstract 5695: Induction of lymphangiogenesis in breast cancer enhances responsiveness to immunotherapies

Abstract Growth and remodeling of tumor-associated lymphatics, termed lymphangiogenesis, has been known to increase metastasis rate in cancer patients. However, a recent study from our group has shown that tumor-associated lymphatics can improve anti-tumor immunity in melanoma upon treatment with immunotherapies. The role of lymphangiogenesis in shaping anti-tumor immune responses in breast cancer, however, remains unexplored. Furthermore, recently approved checkpoint inhibitor immunotherapies have had limited success in breast cancer patients. A better understanding of the role of tumor-associated lymphatics in shaping anti-tumor immune responses could lead to more informed decisions when treating breast cancer patients with immunotherapies. In this study, we investigated the role of induced tumor lymphangiogenesis in modulating responsiveness to different immunotherapies in a murine triple-negative mammary carcinoma model. Spontaneously metastatic 4T1 tumor cells were transduced to over-express the pro-lymphangiogenic growth factor VEGF-C (VC) to induce tumor lymphangiogensis in vivo. Flow cytometry and immunohistochemistry analyses confirmed that VEGF-C over-expressing tumors have an increase in tumor-associated lymphatics than control ones. Higher infiltrations of conv CD4 T cells and macrophages were observed in lymphangiogenic 4T1 tumors. Lymphangiogenic tumors treated with aPD-1 and aCTLA-4 checkpoint inhibitors, or an agonist of the Stimulator of Interferon Genes (STING) pathway, showed improved responsiveness to the therapy compared to the control tumors, as indicated by a reduced tumor growth rate. Moreover, positive correlations between VEGF-C expression and CD4 T cell and macrophage gene expressions were observed in primary human triple-negative breast tumor samples from The Cancer Genome Atlas (TCGA). In conclusion, our study shows that increase in tumor-associated lymphatics in triple-negative breast cancer enhances responsiveness to immunotherapies. Citation Format: Peyman Hosseinchi, Aslan Mansurov, Léa Maillat, Lambert Potin, Gavin Fujimori, Jeffrey A. Hubbell, Melody A. Swartz. Induction of lymphangiogenesis in breast cancer enhances responsiveness to immunotherapies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5695.

Abstract 6141: Role of aldehyde dehydrogenase 1B1 in colon carcinogenesis in a newly developed mouse model

Abstract Introduction: Aldehyde dehydrogenases (ALDH), particularly ALDH1B1 has been shown to have increased expression in both human and APCMin mice colorectal tumors. Studies have proposed the use of ALDH1B1 as a potential novel human colorectal tumor marker. Previous in vitro studies have shown that ALDH1B1 plays a critical role in colon carcinogenesis. The goal of this study is to understand the in vivo role of ALDH1B1 in colon carcinogenesis using a newly developed mouse model, APCMinAldh1b1−/− mice, through characterization of its phenotype in tumor growth and development, and elucidating the underlying potential molecular mechanisms. Methods: We generated APCMinAldh1b1−/-. At 12-14 weeks old, the experimental group APCMinAldh1b1−/- (n=6) and control group APCMinAldh1b1+/+ (n=7) were euthanized. Tumor counts, size and burden for each intestinal segments were recorded. H&amp;E staining was performed and observed for tumor types and counts. Immunohistochemistry using the markers Ki-67 (proliferation), Cleave Caspase 3 (apoptosis), KRT20 (differentiation) were done in colon. IHC of retinoic acid binding proteins (anti-CRBP2 and anti-FABP5) were also performed Results: The experimental group had shown significantly higher total average intestinal tumor burden (6.8 fold) (p=0.0007) and total average tumor counts observed (3.6 fold) (p=0.02). In the colon, there was a 3.5-fold increase in average tumor burden (p=0.017) with a 2.7-fold increase in average tumor size (p=0.025)in the experimental group. We also noticed a trend of increased average tumor counts observed in the colon of the experimental group although statistically insignificant. Average tumor counts of ileum observed was 9-fold higher in experimental group with only 1 mice from control group showed observable small tumor growth which did not meet criteria for tumor size and tumor burden calculation. There were no significant differences in observable tumor counts, tumor size and tumor burden in jejunum. Histologically, all tumors were pedunculated adenocarcinomas. Many unobservable small tumors less than 1mm width and length were seen in the H&amp;E of both ileum and jejunum in the experimental and control group. In the control group, there was less observable tumors compared to the experimental group although the tumor counts on H&amp;E in both groups showed no major differences. This indicates that the tumor size in the experimental group was larger than the control group. On the contrary, average tumor counts of colon in H&amp;E were lower compared to tumor counts observed in both control and experimental groups, indicating low number of small unobservable tumors in colon. Although the tumor counts of colon on H&amp;E and observation between the two groups were statistically insignificant, we noticed a pattern of increased tumor counts in the experimental group suggesting a potential increase in tumor incidence. Immunohistochemical staining of colon is ongoing. Conclusion: ALDH1B1 reduced tumor growth rate in the ileum, and reduced tumor size in the colon suggesting its role in tumor promotion and progression. A larger number of experimental and control group is required to establish its potentially tumor-initiating role in the colon. Further characterization of ALDH1B1 in colon carcinogenesis is ongoing. We are also elucidating the role and regulation of retinoic acid signaling by ALDH1B1 in colorectal tumor growth. Citation Format: Wan Ying Tan, David. J. Orlicky, Ying Chen, Yatrik M. Shah, David C. Thompson, Vasilis Vasiliou. Role of aldehyde dehydrogenase 1B1 in colon carcinogenesis in a newly developed mouse model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6141.

Abstract 4717: Ly6-neurotoxin1 knockout in PDAC cells inhibits their growth in vitro and in vivo

Abstract Ly6/neurotoxin1 (Lynx1) functions as a brake for nicotinic receptors and was defined as a tumor suppressor in lung cancer. As pancreatic cancer development may be slowed down by cholinergic signaling, we investigated the role of Lynx1 in pancreatic ductal adenocarcinoma (PDAC) cell lines, both in vitro and in vivo. Lynx1 knockout cell clones were generated by transfecting the CRISPRCas9 plasmid - (pSpCas9 (BB)-2A-Puro) into Miapacaluci and BXPC3luci PDAC cells using jet Pei and jet Prime as transfecting agents, respectively. The annealed gRNA was directed towards exon 3 of the Lynx1 gene. Control clones were transfected with the plasmid alone. Clones from both cell lines were tested by qRT-PCR and Western blot for knockout efficiency as well as by proliferation assay. In addition, the expression levels of p-MEK, p-MAPK, p-mTOR and p-Rictor were verified in two BXPC3 cell clones in relation to Lynx1 expression. Furthermore, 4x106 cells of four BXPC3 cell clones (one control, 3 knockout clones) were injected, respectively, into the portal vein of nude rats to control for a possibly reduced tumor growth in the liver. The knockdown of Lynx1 was incomplete at mRNA level and ranged from 20 to 60% in BXPC3 clones and from 0 to 30% in Miapaca clones. At protein level, the respective values ranged from 0 to 90% in BXPC3 and from 22 to 50% in Miapaca clones. However, Lynx1 protein levels increased at later time points. All BXPC3 clones proliferated less quickly than the respective control when tested by MTT assay. Miapaca clones, however, didn't show a significant difference from the respective control, although their growth was clearly disturbed shortly after transfection. Concomitantly with reduced Lynx1 protein levels, there was reduction of p-mTOR (90%), p-Rictor (30%) and p-MEK (40%) in BXPC3 cells. In vivo, the BXPC3 clones showed a lag period of 1 to 2 weeks till the appearance of a first bioluminescence signal indicating tumor growth. Rats injected with cells from the control clone showed a steady increase in the bioluminescence signal (n=7 of 10) as compared to the most sensitive knockdown clone, which didn't show any signal in any of 4 injected rats (p=0.05). Cells of 2 other BXPC3 clones showed a reduced growth rate at best in 2 of 2 rats used for each clone, respectively. In conclusion, knockout of Lynx1 was incomplete at both mRNA and protein levels. Nevertheless, the respective BXPC3 clones exhibited reduced proliferation in vitro, which was associated with diminished p-mTOR, p-Rictor and p-MEK levels. In addition, they failed to establish a tumor in vivo or showed a reduced tumor growth rate. These findings suggest that Lynx1 is a vital gene and may play an important role in the growth and establishment of PDAC cells. Citation Format: Doaa Ali, Michael Zepp, Matthias Bozza, Maria Nikolova, Richard Harbottle, Martin R. Berger. Ly6-neurotoxin1 knockout in PDAC cells inhibits their growth in vitro and in vivo [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4717.