Overall Survival Of Tumor-bearing Mice Research Articles

Tumor-Specific Protein Induced in Situ Self-Assembly of Peptide Drugs for Synergistic Mitochondria Disruption.

Mitochondria-targeted cancer therapy is an effective method for controlling tumor growth. However, the presence of repair mechanisms in tumor cells in response to mitochondrial damage poses significant challenges for treatment. By taking advantage of intracellular self-assembly technology, a peptide nanomaterial, RC-K-FX, that enters tumor cells in a monomeric form is designed. After binding to MUC1-C inside the cell membrane, RC-K-FX assembles into a spherical structure that stably encapsulates MUC1-C, inhibiting its dimerization and blocking the repair of stress-induced mitochondrial damage in tumor cells. Moreover, the self-assembled mitochondrial toxic peptide effectively destroys the mitochondria, and the loss of mitochondrial repair significantly increases tumor cytotoxicity by disrupting the redox balance, enhancing reactive oxygen species (ROS), inhibiting the nuclear factor (NF)-κB pathway, and suppressing the epithelial-mesenchymal transition (EMT) process. After intravenous administration, RC-G-FX accumulated at the tumor site, exhibiting improved anti-tumor effects and extending the overall survival of tumor-bearing mice. Therefore, the integration of the in situ self-assembly of peptide drugs and damage to mitochondrial repair mechanisms provides effective therapeutic options for malignancy.

EXTH-21. WINNING THE TUG OF WAR BETWEEN VIRUS AND TUMOR IMMUNODOMINANCE IN GLIOMAS TREATED WITH ONCOLYTIC ADENOVIRUSES USING TOLEROGENIC STRATEGIES

Abstract Phase 1 and 2 clinical trials of our oncolytic adenovirus Delta-24-RGD for patients with recurrent malignant gliomas have demonstrated its safety and efficacy, and showed that 20% of the patients experienced long-term tumor remissions often associated with inflammatory responses and tumor infiltration of lymphocytes. Based on our preliminary data, we expected that the most frequent tumor-infiltrating CD8+ T cell clones will target adenoviral hexon, with a single virus-specific clone representing up to 5% of the total population. Competition between T cells for resources may limit the expansion of subdominant clones recognizing tumor antigens. We hypothesized that mitigating virus-specific immunity allows the expansion of tumor-specific T cells and results in better therapeutic efficacy. In mice, intravenously injected nanoparticles encapsulating adenoviral antigens preferentially accumulated in the liver (P<0.0001), where self-reactive immune cells are deleted through peripheral tolerance. RNA-sequencing of antigen-specific T cells isolated after nanoparticle injection revealed enriched expression of gene sets involved in immune tolerance through clonal deletion and T cell anergy, including increased expression of immune checkpoint molecules (P<0.001). In glioma-bearing mice treated with virotherapy in combination with nanoparticle administration, tumor infiltrating lymphocytes exhibited decreased IFNγ secretion against viral antigens and increased secretion against tumor antigens, suggesting a redirection of the immunity from anti-viral to anti-tumoral (P<0.05). Importantly, virotherapy with nanoparticles increased the overall survival of tumor-bearing mice compared to virus treatment alone (P<0.001). To further characterize the roles of virus- and tumor-specific immune cells during virotherapy we are analyzing the T cell receptor repertoire of surgical specimens from nine glioma patients treated with Delta-24-RGD in a phase 1 clinical trial. In summary, our data provide the basis for a future clinical trial using tolerogenic strategies to redirect immunodominance of the viral to the glioma antigens, and represent a novel strategy to enhance anti-tumor immunity during virotherapy of brain tumors.

Inhibiting NR5A2 targets stemness in pancreatic cancer by disrupting SOX2/MYC signaling and restoring chemosensitivity

BackgroundPancreatic ductal adenocarcinoma (PDAC) is a profoundly aggressive and fatal cancer. One of the key factors defining its aggressiveness and resilience against chemotherapy is the existence of cancer stem cells (CSCs). The important task of discovering upstream regulators of stemness that are amenable for targeting in PDAC is essential for the advancement of more potent therapeutic approaches. In this study, we sought to elucidate the function of the nuclear receptor subfamily 5, group A, member 2 (NR5A2) in the context of pancreatic CSCs.MethodsWe modeled human PDAC using primary PDAC cells and CSC-enriched sphere cultures. NR5A2 was genetically silenced or inhibited with Cpd3. Assays included RNA-seq, sphere/colony formation, cell viability/toxicity, real-time PCR, western blot, immunofluorescence, ChIP, CUT&Tag, XF Analysis, lactate production, and in vivo tumorigenicity assays. PDAC models from 18 patients were treated with Cpd3-loaded nanocarriers.ResultsOur findings demonstrate that NR5A2 plays a dual role in PDAC. In differentiated cancer cells, NR5A2 promotes cell proliferation by inhibiting CDKN1A. On the other hand, in the CSC population, NR5A2 enhances stemness by upregulating SOX2 through direct binding to its promotor/enhancer region. Additionally, NR5A2 suppresses MYC, leading to the activation of the mitochondrial biogenesis factor PPARGC1A and a shift in metabolism towards oxidative phosphorylation, which is a crucial feature of stemness in PDAC. Importantly, our study shows that the specific NR5A2 inhibitor, Cpd3, sensitizes a significant fraction of PDAC models derived from 18 patients to standard chemotherapy. This treatment approach results in durable remissions and long-term survival. Furthermore, we demonstrate that the expression levels of NR5A2/SOX2 can predict the response to treatment.ConclusionsThe findings of our study highlight the cell context-dependent effects of NR5A2 in PDAC. We have identified a novel pharmacological strategy to modulate SOX2 and MYC levels, which disrupts stemness and prevents relapse in this deadly disease. These insights provide valuable information for the development of targeted therapies for PDAC, offering new hope for improved patient outcomes.Graphical A Schematic illustration of the role of NR5A2 in cancer stem cells versus differentiated cancer cells, along with the action of the NR5A2 inhibitor Cpd3. B Overall survival of tumor-bearing mice following allocated treatment. A total of 18 PDX models were treated using a 2 x 1 x 1 approach (two animals per model per treatment); n=36 per group (illustration created with biorender.com).

P02.20.B PLASMA MEMBRANE-ASSOCIATED 70 KDA HEAT SHOCK PROTEIN (HSP70) INVOLVED IN GLIOBLASTOMA CELLS INVASION AS A TARGET FOR TUMOR THERANOSTICS

Abstract BACKGROUND Heat shock proteins (HSPs) represent a large family of highly conserved proteins serving as molecular chaperones that play an important role in intracellular homeostasis. Apart from their intracellular localization, members of various HSPs families such as HSP70 have been reported to be expressed on the plasma membrane of malignant cells, including glioblastoma multiforme (GBM). However, the role of the membrane-bound protein Hsp70 has not yet been investigated in the pathophysiology of GBM. MATERIAL AND METHODS mHsp70 expression as related to GBM cell invasive potential was analyzed in biopsies obtained from neurooncological patients using CellVoyager CQ1 Benchtop High-Content Analysis System (Yokogawa) and inverted confocal microscopy (Leica Microsystems). Application of membrane-associated chaperone for targeted therapies was assessed in the intracranial human NCH644, NCH421k, and U87 GBM models in immunodeficient mice employing various chaperone inhibitors (i.e., JG-98, MKT-077, pifithrin-μ (PES)) as a monotherapy or in combination with a single dose of stereotactic irradiation (10Gy) (SARRP). RESULTS Live-cell imaging of the patient-derived biopsies demonstrated the increased cellular density of mHsp70+ GBM cells particularly in the tissue along the tumor-brain margin. Time-lapse evaluation of the tumor sample further proved the migration of mHsp70+ cells into the surrounding normal brain tissues. Subsequent high-powered multiplex immunofluorescence analysis (Akoya Biosystems) confirmed significantly increased single-cell infiltration of mHsp70+ tumor cells (also positively stained for Nestin, Oct4, and SOX2) in the normal brain tissues. When mHsp70 inhibitors were applied in GBM animal models as a monotherapy or in combination with radiotherapy dramatic inhibition of tumor progression (as shown by MRI) and increased animal survival were observed. CONCLUSION Expression of the mHsp70 on the plasma membrane of GBM cells corresponds to the highly invasive potential of the tumor cells in neuro-oncological patients. Administration of the HSP70 small molecular inhibitors significantly decreased the GBM growth and invasion thus prolonging the overall survival of tumor-bearing mice. Further development of mHsp70-targeted agents represents a promising approach in treatment of multiforme glioblastoma.

Recombinant human cyclophilin A in combination with adoptive T cell therapy improves the efficacy of cancer immunotherapy in experimental models in vivot;

Adoptive T-cell therapy (ACT) is successfully applied in cancer treatment, however, its efficiency can be limited by a low viability, short persistence time, and loss of functional activity of T cells after adoptive transfer. The search for novel immunomodulators that can improve the viability, expansion, and functions of T cells after their infusion with the minimal side effects could contribute to the development of more efficient and safe ACT strategies. Recombinant human cyclophilin A (rhCypA) is of particular interest in this respect, as it exhibits pleiotropic immunomodulatory activity and stimulates both innate and adaptive anti-tumor immunity. Here, we evaluated the effect of rhCypA on the efficacy of ACT in the mouse EL4 lymphoma model. Lymphocytes from transgenic 1D1a mice with an inborn pool of EL4-specific T cells were used as a source of tumor-specific T cells for ACT. In models of immunocompetent and immunodeficient transgenic mice, the course (3 days) rhCypA administration was shown to significantly stimulate EL4 rejection and prolong the overall survival of tumor-bearing mice after adoptive transfer of lowered doses of transgenic 1D1a cells. Our studies showed that rhCypA significantly improved the efficacy of ACT by enhancing the effector functions of tumor-specific cytotoxic T cells. These findings open up the prospects for the development of innovative strategies of adoptive T-cell immunotherapy for cancer using rhCypA as an alternative to existing cytokine therapies.

Breast Cancer Stem Cell-Derived Tumors Escape from γδ T-cell Immunosurveillance In Vivo by Modulating γδ T-cell Ligands.

There are no targeted therapies for patients with triple-negative breast cancer (TNBC). TNBC is enriched in breast cancer stem cells (BCSC), which play a key role in metastasis, chemoresistance, relapse, and mortality. γδ T cells hold great potential in immunotherapy against cancer and might provide an approach to therapeutically target TNBC. γδ T cells are commonly observed to infiltrate solid tumors and have an extensive repertoire of tumor-sensing mechanisms, recognizing stress-induced molecules and phosphoantigens (pAgs) on transformed cells. Herein, we show that patient-derived triple-negative BCSCs are efficiently recognized and killed by ex vivo expanded γδ T cells from healthy donors. Orthotopically xenografted BCSCs, however, were refractory to γδ T-cell immunotherapy. We unraveled concerted differentiation and immune escape mechanisms: xenografted BCSCs lost stemness, expression of γδ T-cell ligands, adhesion molecules, and pAgs, thereby evading immune recognition by γδ T cells. Indeed, neither promigratory engineered γδ T cells, nor anti-PD-1 checkpoint blockade, significantly prolonged overall survival of tumor-bearing mice. BCSC immune escape was independent of the immune pressure exerted by the γδ T cells and could be pharmacologically reverted by zoledronate or IFNα treatment. These results pave the way for novel combinatorial immunotherapies for TNBC.

Functional characterization of age-dependent p16 epimutation reveals biological drivers and therapeutic targets for colorectal cancer

BackgroundMethylation of the p16 promoter resulting in epigenetic gene silencing—known as p16 epimutation—is frequently found in human colorectal cancer and is also common in normal-appearing colonic mucosa of aging individuals. Thus, to improve clinical care of colorectal cancer (CRC) patients, we explored the role of age-related p16 epimutation in intestinal tumorigenesis.MethodsWe established a mouse model that replicates two common genetic and epigenetic events observed in human CRCs: Apc mutation and p16 epimutation. We conducted long-term survival and histological analysis of tumor development and progression. Colonic epithelial cells and tumors were collected from mice and analyzed by RNA sequencing (RNA-seq), quantitative PCR, and flow cytometry. We performed single-cell RNA sequencing (scRNA-seq) to characterize tumor-infiltrating immune cells throughout tumor progression. We tested whether anti-PD-L1 immunotherapy affects overall survival of tumor-bearing mice and whether inhibition of both epigenetic regulation and immune checkpoint is more efficacious.ResultsMice carrying combined Apc mutation and p16 epimutation had significantly shortened survival and increased tumor growth compared to those with Apc mutation only. Intriguingly, colon tumors with p16 epimutation exhibited an activated interferon pathway, increased expression of programmed death-ligand 1 (Pdl1), and enhanced infiltration of immune cells. scRNA-seq further revealed the presence of Foxp3+ Tregs and γδT17 cells, which contribute to an immunosuppressive tumor microenvironment (TME). Furthermore, we showed that a combined therapy using an inhibitor of DNA methylation and a PD-L1 immune checkpoint inhibitor is more effective for improving survival in tumor-bearing mice than blockade of either pathway alone.ConclusionsOur study demonstrated that age-dependent p16 epimutation creates a permissive microenvironment for malignant transformation of polyps to colon cancer. Our findings provide a mechanistic rationale for future targeted therapy in patients with p16 epimutation.

Eubacterium rectale Improves the Efficacy of Anti-PD1 Immunotherapy in Melanoma via l-Serine-Mediated NK Cell Activation.

Natural killer (NK) cells, as key immune cells, play essential roles in tumor cell immune escape and immunotherapy. Accumulating evidence has demonstrated that the gut microbiota community affects the efficacy of anti-PD1 immunotherapy and that remodeling the gut microbiota is a promising strategy to enhance anti-PD1 immunotherapy responsiveness in advanced melanoma patients; however, the details of the mechanism remain elusive. In this study, we found that Eubacterium rectale was significantly enriched in melanoma patients who responded to anti-PD1 immunotherapy and that a high E. rectale abundance was related to longer survival in melanoma patients. Furthermore, administration of E. rectale remarkably improved the efficacy of anti-PD1 therapy and increased the overall survival of tumor-bearing mice; moreover, application of E. rectale led to a significant accumulation of NK cells in the tumor microenvironment. Interestingly, conditioned medium isolated from an E. rectale culture system dramatically enhanced NK cell function. Gas chromatography-mass spectrometry/ultrahigh performance liquid chromatography-tandem mass spectrometry-based metabolomic analysis showed that l-serine production was significantly decreased in the E. rectale group; moreover, administration of an l-serine synthesis inhibitor dramatically increased NK cell activation, which enhanced anti-PD1 immunotherapy effects. Mechanistically, supplementation with l-serine or application of an l-serine synthesis inhibitor affected NK cell activation through Fos/Fosl. In summary, our findings reveal the role of bacteria-modulated serine metabolic signaling in NK cell activation and provide a novel therapeutic strategy to improve the efficacy of anti-PD1 immunotherapy in melanoma.

Single cell sequencing reveals that CD39 inhibition mediates changes to the tumor microenvironment

Single-cell sequencing technologies have noteworthily improved our understanding of the genetic map and molecular characteristics of bladder cancer (BC). Here we identify CD39 as a potential therapeutic target for BC via single-cell transcriptome analysis. In a subcutaneous tumor model and orthotopic bladder cancer model, inhibition of CD39 (CD39i) by sodium polyoxotungstate is able to limit the growth of BC and improve the overall survival of tumor-bearing mice. Via single cell RNA sequencing, we find that CD39i increase the intratumor NK cells, conventional type 1 dendritic cells (cDC1) and CD8 + T cells and decrease the Treg abundance. The antitumor effect and reprogramming of the tumor microenvironment are blockaded in both the NK cells depletion model and the cDC1-deficient Batf3−/− model. In addition, a significant synergistic effect is observed between CD39i and cisplatin, but the CD39i + anti-PD-L1 (or anti-PD1) strategy does not show any synergistic effects in the BC model. Our results confirm that CD39 is a potential target for the immune therapy of BC.

Chimeric Antigen Receptor T Cells With Modified Interleukin-13 Preferentially Recognize IL13Rα2 and Suppress Malignant Glioma: A Preclinical Study.

BackgroundInterleukin-13 receptor α 2 (IL13Rα2) is a promising tumor-directed antigen of malignant glioma (MG). Here, we examine the efficacy and safety of T cells containing a YYB-103 chimeric antigen receptor (CAR) that can preferentially bind to IL13Rα2 on MG cells.MethodsIL13 was modified on the extracellular domain by substitution of amino acids with E13K, R66D, S69D, and R109K and stably transfected into human T cells using a retroviral vector. The in vitro efficacy of YYB-103 CAR T cells was tested in cell lines with differing IL13Rα1 and IL13Rα2 expression. The in vivo efficacy of intracerebroventricular (i.c.v.) and intravenous (i.v.) routes of YYB-103 CAR T-cell administration were tested in orthotopic MG mouse models. Immunohistochemical staining of MG was performed using WHO grade 3/4 surgical specimens from 53 patients. IL13Rα2 expression was quantified by H-score calculated from staining intensity and percentage of positive cells.ResultsBinding affinity assay of YYB-103 verified apparently nil binding to IL13Rα1, which was more selective than previously reported IL13 modification (E13Y). YYB-103 CAR T cells showed selective toxicity toward co-cultured U87MG (IL13Rα1+/IL13Rα2+) cells but not A431 (IL13Rα1+/IL13Rα2−) cells. Consistently, YYB-103 CAR T cells suppressed tumor growth in nude mice receiving orthotopic injection of U87 MG cells. Both i.c.v. and i.v. injections of YYB-103 CAR T cells reduced tumor volume and prolonged overall survival of tumor-bearing mice. The median H-score for IL13Rα2 in patient-derived MG tissue was 5 (mean, 57.5; SD, 87.2; range, 0 to 300).ConclusionThis preclinical study demonstrates the efficacy of IL13Rα2-targeted YYB-103 CAR T cells against MG cells. The use of modified IL13 to construct a CAR facilitated the selective targeting of IL13Rα2-expressing MG cells while sparing IL13Rα1-expressing cells. Notably, YYB-103 CAR T cells exhibited effective blood–brain barrier crossing, suggesting compatibility with i.v. administration rather than intracranial injection. Additionally, the high H-score for IL13Rα2 in glioblastoma, especially in conjunction with the poor prognostic markers of wild-type isocitrate dehydrogenase-1 (IDH-1) and unmethylated O 6-methyl guanine methyl-transferase (MGMT), could be used to determine the eligibility of patients with recurrent glioblastoma for a future clinical trial of YYB-103 CAR T cells.

Myofibroblast-Specific Msi2 Knockout Inhibits HCC Progression in a Mouse Model.

Myofibroblasts play a pivotal role in the development and progression of HCC. Here, we aimed to explore the role and mechanism of myofibroblast Musashi RNA binding protein 2 (MSI2) in HCC progression. Myofibroblast infiltration and collagen deposition were detected and assessed in the tissues from 117 patients with HCC. Transgenic mice (Msi2ΔCol1a1 ) with floxed Msi2 allele and collagen type I alpha 1 chain (Col1a1)-ligand inducible Cre recombinases (CreER) were constructed to generate a myofibroblast-specific Msi2 knockout model. Mouse HCC cells were orthotopically transplanted into the Msi2ΔCol1a1 or the control mice (Msi2F/F ). We found that the deposition of collagen fibers, the main product of myofibroblasts, predicted a poor prognosis for HCC; meanwhile, we detected high MSI2 expression in the peritumoral infiltrated myofibroblasts. Conditional deletion of Msi2 in myofibroblasts significantly inhibited the growth of orthotopically implanted HCC, reduced both intrahepatic and lung metastasis, and prolonged the overall survival of tumor-bearing mice (P=0.002). In vitro analysis demonstrated that myofibroblasts promoted cell proliferation, invasion, and epithelial-mesenchymal transformation of HCC cells, whereas Msi2 deletion in myofibroblasts reversed these effects. Mechanically, Msi2 knockout decreased myofibroblast-derived IL-6 and IL-11 secretion by inhibiting the extracellular signal-regulated kinase 1/2 pathway, and thus attenuated the cancer stem cell-promoting effect of myofibroblasts. Interestingly, we found that the simultaneous knockout of Msi2 in myofibroblasts and knockdown of Msi2 in HCC cells could not further attenuate the implanted HCC progression. Myofibroblast-specific Msi2 knockout abrogated the tumor-promoting function of myofibroblasts and inhibited HCC progression in mouse models. Targeting myofibroblast MSI2 expression may therefore prove to be a therapeutic strategy for HCC treatment in the future.

Systemic metastasis-targeted nanotherapeutic reinforces tumor surgical resection and chemotherapy

Failure of conventional clinical therapies such as tumor resection and chemotherapy are mainly due to the ineffective control of tumor metastasis. Metastasis consists of three steps: (i) tumor cells extravasate from the primary sites into the circulation system via epithelial-mesenchymal transition (EMT), (ii) the circulating tumor cells (CTCs) form “micro-thrombi” with platelets to evade the immune surveillance in circulation, and (iii) the CTCs colonize in the pre-metastatic niche. Here, we design a systemic metastasis-targeted nanotherapeutic (H@CaPP) composed of an anti-inflammatory agent, piceatannol, and an anti-thrombotic agent, low molecular weight heparin, to hinder the multiple steps of tumor metastasis. H@CaPP is found efficiently impeded EMT, inhibited the formation of “micro-thrombi”, and prevented the development of pre-metastatic niche. When combined with surgical resection or chemotherapy, H@CaPP efficiently inhibits tumor metastasis and prolonged overall survival of tumor-bearing mice. Collectively, we provide a simple and effective systemic metastasis-targeted nanotherapeutic for combating tumor metastasis.

Co-delivery of IL-12 cytokine gene and cisplatin prodrug by a polymetformin-conjugated nanosystem for lung cancer chemo-gene treatment through chemotherapy sensitization and tumor microenvironment modulation

The combination of chemotherapy and gene therapy has been indicated as a promising approach for cancer therapy. However, this combination strategy is still faced a challenge by the lack of suitable carriers to co-loaded chemotherapeutic drug and gene into one single nanoplatform. In this study, a tumor-targeted HC/pIL-12/polyMET micelleplexes were developed for the co-loading and co-delivery of cisplatin (CDDP) and plasmid encoding interleukin-12 gene (pIL-12), which would be utilized to generate synergistic actions through chemotherapy sensitization and microenvironment modulation. The HC/pIL-12/polyMET exhibited desirable particle size, superior serum stability, effective intracellular CDDP release and pIL-12 transfection efficiency. More important, the HC/pIL-12/polyMET generated the enhanced LLC cell proliferation inhibition and apoptosis induction efficiency. The long-circulating HC/pIL-12/polyMET micelleplexes promoted the accumulation of CDDP and pIL-12 in tumor site, which resulted in significantly inhibiting the growth of lung cancer, and prolonging the overall survival of tumor-bearing mice. The underlying immune mechanism demonstrated the combination of CDDP and pIL-12 activated immune effector cells to release IFN-γ and induced M1-type differentiation of tumor-related macrophages, thereby generating synergistic chemoimmunotherapy effect. Taken together, this study may provide an effective strategy for drug/gene co-delivery and cancer chemoimmunotherapy. Statement of significanceChemoimmunotherapy has been indicated as an approach to improve efficacy of cancer therapy. Herein, a tumor-targeted micelleplexes (HC/pIL-12/polyMET) were developed for the co-delivery of cisplatin (CDDP) and plasmid encoding IL-12 gene (pIL-12), which can employ the synergistic effects through chemotherapy sensitization and microenvironment modulation. The HC/pIL-12/polyMET exhibited desirable particle size, superior serum stability, high gene transfection efficiency and antitumor activity on tumor cell proliferation inhibition and apoptosis induction. More importantly, the long-circulating HC/pIL-12/polyMET micelleplexes could effectively accumulate in tumor sites and then rapidly release the CDDP and pIL-12, significantly inhibit the growth of lung cancer. This strategy provides a new concept for chemo-gene combination with a strengthened overall therapeutic efficacy of chemoimmunotherapy.

Fascin inhibitor increases intratumoral dendritic cell activation and anti-cancer immunity

SUMMARYFascin protein is the main actin-bundling protein in filopodia and invadopodia, which are critical for tumor cell migration, invasion, and metastasis. Small-molecule fascin inhibitors block tumor invasion and metastasis and increase the overall survival of tumor-bearing mice. Here, we report a finding that fascin blockade additionally reinvigorates anti-tumor immune response in syngeneic mouse models of various cancers. Fascin protein levels are increased in conventional dendritic cells (cDCs) in the tumor microenvironment. Mechanistically, fascin inhibitor NP-G2–044 increases the number of intratumoral-activated cDCs and enhances the antigen uptake by cDCs. Furthermore, together with PD-1 blocking antibody, NP-G2–044 markedly increases the number of activated CD8+ T cells in the otherwise anti-PD-1 refractory tumors. Reduction of fascin levels in cDCs, but not fascin gene knockout in tumor cells, mimics the anti-tumor immune effect of NP-G2–044. These data demonstrate that fascin inhibitor NP-G2–044 simultaneously limits tumor metastasis and reinvigorates anti-tumor immune responses.

AP-2α Regulates S-Phase and Is a Marker for Sensitivity to PI3K Inhibitor Buparlisib in Colon Cancer.

Activating protein 2 alpha (AP-2α; encoded by TFAP2A) functions as a tumor suppressor and influences response to therapy in several cancer types. We aimed to characterize regulation of the transcriptome by AP-2α in colon cancer. CRISPR-Cas9 and short hairpin RNA were used to eliminate TFAP2A expression in HCT116 and a panel of colon cancer cell lines. AP-2α target genes were identified with RNA sequencing and chromatin immunoprecipitation sequencing. Effects on cell cycle were characterized in cells synchronized with aphidicolin and analyzed by FACS and Premo FUCCI. Effects on invasion and tumorigenesis were determined by invasion assay, growth of xenografts, and phosphorylated histone H3 (PHH3). Knockout of TFAP2A induced significant alterations in the transcriptome including repression of TGM2, identified as a primary gene target of AP-2α. Loss of AP-2α delayed progression through S-phase into G2-M and decreased phosphorylation of AKT, effects that were mediated through regulation of TGM2. Buparlisib (BKM120) repressed in vitro invasiveness of HCT116 and a panel of colon cancer cell lines; however, loss of AP-2α induced resistance to buparlisib. Similarly, buparlisib repressed PHH3 and growth of tumor xenografts and increased overall survival of tumor-bearing mice, whereas, loss of AP-2α induced resistance to the effect of PI3K inhibition. Loss of AP-2α in colon cancer leads to prolonged S-phase through altered activation of AKT leading to resistance to the PI3K inhibitor, Buparlisib. The findings demonstrate an important role for AP-2α in regulating progression through the cell cycle and indicates that AP-2α is a marker for response to PI3K inhibitors. IMPLICATIONS: AP-2α regulated cell cycle through the PI3K cascade and activation of AKT mediated through TGM2. AP-2α induced sensitivity to Buparlisib/BKM120, indicating that AP-2α is a biomarker predictive of response to PI3K inhibitors.

Enhanced brain delivery and therapeutic activity of trastuzumab after blood-brain barrier opening by NEO100 in mouse models of brain-metastatic breast cancer.

The antitumor efficacy of human epidermal growth factor receptor 2 (HER2)-targeted therapies, such as humanized monoclonal antibody trastuzumab (Herceptin®, Roche), in patients with breast-to-brain cancer metastasis is hindered by the low permeability of the blood-brain barrier (BBB). NEO100 is a high-purity version of the natural monoterpene perillyl alcohol, produced under current good manufacturing practice (cGMP) regulations, that was shown previously to reversibly open the BBB in rodent models. Here we investigated whether NEO100 could enable brain entry of trastuzumab to achieve greater therapeutic activity. An in vitro BBB, consisting of human astrocytes and brain endothelial cells, was used to determine trastuzumab penetration in the presence or absence of NEO100. For in vivo studies, we administered intravenous (IV) trastuzumab or the trastuzumab-drug conjugate ado-trastuzumab emtansine (T-DM1; Kadcyla®, Roche), to mouse models harboring intracranial HER2+ breast cancer, with or without BBB opening via IA NEO100. Brain and tumor tissues were examined for the presence of trastuzumab and infiltration of immune cells. Therapeutic impact was evaluated based on overall survival. NEO100 greatly increased trastuzumab penetration across an in vitro BBB. In vivo, IA NEO100-mediated BBB opening resulted in brain tumor-selective accumulation of trastuzumab, without detectable presence in normal brain tissue, along with increased presence of immune cell populations. IV delivery of trastuzumab or T-DM1 achieved significantly greater overall survival of tumor-bearing mice when combined with IA NEO100. IA NEO100 facilitates brain tumor entry of trastuzumab and T-DM1 and significantly enhances their therapeutic efficacy, along with increased antibody-dependent immune cell recruitment.

'Off-the-shelf’ allogeneic antigen-specific adoptive T-cell therapy for the treatment of multiple EBV-associated malignancies

BackgroundEpstein-Barr virus (EBV), an oncogenic human gammaherpesvirus, is associated with a wide range of human malignancies of epithelial and B-cell origin. Recent studies have demonstrated promising safety and clinical efficacy...

Gemcitabine and Rapamycin Exhibit Additive Effect against Osteosarcoma by Targeting Autophagy and Apoptosis.

Simple SummaryOsteosarcoma is the most common solid cancer of the bone. Unfortunately, the expected outcome for patients with this and similar types of cancers has changed little over the last 20 years. One important need is to develop new therapies that overcome the development of drug resistance in osteosarcoma cells. The drug gemcitabine (Gem) can kill osteosarcoma cells and prevent them from growing and spreading. Another drug, rapamycin (Rapa), can also kill osteosarcoma cells, but does so in a different way. Here, we investigated whether a combination of Rapa and Gem would exhibit better osteosarcoma treatment efficiency than Rapa and Gem monotherapies. We found that this drug combination was very effective at killing osteosarcoma cells, both in cell cultures and in mice, and the combinatorial treatment was better than treatment with a single agent. Therefore, combinatorial therapy with Gem and Rapa may be an effective approach for treating drug resistant osteosarcoma cells.The overall prognosis for sarcoma-based cancer patients has remained largely unchanged over the past 10 years. Because there is no effective anticancer drug for patients with chemoresistant osteosarcoma (OS), novel approaches are needed to improve the prognosis. Here, we investigated whether rapamycin (Rapa) could enhance the anti-tumor effects of gemcitabine (Gem) in OS. Gem dose-dependently killed the OS cells, but exhibited much lower cytotoxicity on osteoblasts. Treatment with a combination Gem and Rapa was much more effective than that of either single agent with respect to reducing cell viability, cell invasion, cell migration, and vascular endothelial growth factor production in vitro. Moreover, the combination of these agents suppressed tumor growth, angiogenesis, and lung metastasis in allograft and xenograft murine models of OS with minimal adverse effects. Overall, the combination therapy prolonged the overall survival of tumor-bearing mice. Mechanistically, Gem induced apoptosis and increased the levels of cleaved caspases, while Rapa induced autophagy and microtubule-associated protein light chain 3 (LC3)-I/LC3-II expression both in vitro and in vivo. Our findings suggest that chemotherapy using Gem combined with Rapa may be a novel and promising therapeutic approach for the treatment of OS.

Abstract 6257: DPX™ platform for drug delivery: Evaluation of lymph node-targeted delivery of immunomodulatory agents in DPX

Abstract Small molecule drugs and biological macromolecules are currently being investigated as therapies for various types of disorders such as cancer and infectious disease. Generally, these agents are systemically administered. However, many immunomodulatory agents are active primarily on specific immune cells such as dendritic cells or T cells. The purpose of this study was to evaluate the efficacy of the novel delivery platform, DPX, for targeted delivery of immunomodulatory agents to the lymph node. As the lymph node is the primary site for priming and activation of immune cells, this approach may improve delivery over systemic administration resulting in increased drug efficacy, the use of less drug, and reduced off-target toxicity. To evaluate the efficacy of the DPX platform for lymph node-targeted drug delivery, we used the C57Bl/6 murine model. DPX formulation of small molecule dyes allowed us to visualize the kinetics of lymph node-specific delivery by visual assessments and by flow cytometry. DPX-dye formulations demonstrated a controlled and sustained uptake of molecules from the site of injection, travelling to the regional lymph node, without significant systemic exposure. To test impacts on tumor directed immune responses, the HPV16 E7 expressing C3 tumor model was used. Novel formulations were tested in conjunction with DPX-FP (a C3 tumor-specific antigen HPV16 E749-57 conjugated to a T helper epitope). DPX-mediated delivery of low doses of cyclophosphamide (CPA) were delivered subcutaneously as a single injection. This low dose of CPA has shown to act as an immunomodulatory agent when delivered systemically over a week. Using DPX to deliver CPA resulted in a significant reduction in the number of lymph node cells. This was evident after a single administration, showing equivalent therapeutic benefit was achieved with significantly less active agent. This was observed irrespective of whether CPA was administered alone or with DPX-FP. We also evaluated DPX delivery of the checkpoint inhibitor anti-CTLA-4. We found that administration in DPX was equivalent to systemic administration by intraperitoneal injection in terms of overall survival of tumor-bearing mice and in the proportion of CD3+ T cells bound by anti-CTLA-4, with fewer administrations of antibody. This demonstrates maintenance of biological functionality of macromolecules in the DPX platform. In conclusion, DPX holds promise in terms of lymph node-targeted delivery of functional agents. This delivery is facilitated by a sustained uptake from the site of injection and can offer reduced dosing and off-target toxicity as compared to systemic delivery. Many approaches to targeted therapies have failed to provide a therapeutic index commensurate with their promise through systemic delivery, with a challenge in achieving selectivity for the target cell type. Here, we show the DPX drug delivery platform has the potential to overcome this challenge and thereby increase the efficacy of immune and cancer therapies. Citation Format: Heather Torrey, Ava Vila-Leahey, Yuchen Cen, Danielle MacKay, Alecia MacKay, Leanne Lucas, Brittany Myers, Arthvan Sharma, Douglas Vieira, Andrea Penwell, Rajkannan Rajagopalan, Marianne Stanford. DPX™ platform for drug delivery: Evaluation of lymph node-targeted delivery of immunomodulatory agents in DPX [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 6257.

Abstract 1110: A novel peptide based microRNA nanoparticle hydrogel composite attenuates mesothelioma growth

Abstract Malignant pleural mesothelioma (MPM) is an aggressive cancer resistant to multimodal therapies. Current therapeutic drug regimens ultimately fail due to a poor understanding of the pathogenic mechanisms. After surgical resection, MPM inevitably recurs from residual microscopic tumor foci. These clinical challenges led us to design a locoregional therapeutic platform delivering microRNA (miRNA), a novel anti-cancer agent, intrapleurally, with the goal of eradicating residual microscopic tumor foci thereby improving the efficacy of multimodal therapy. We developed a peptide-based biodegradable hydrogel that can be injected or sprayed directly to coat anatomic surfaces, functioning as a therapeutic depot. Properties of the hydrogel were fine-tuned for biopersistence time, therapeutic payload, cell-specific targeting etc. In the first step of formulation, miRNA complexed with an amphiphilic cationic peptide forms stable nanoparticles. Next, miRNA-nanoparticles were encapsulated into another peptide hydrogel, formed via self-assembly of amphiphilic cationic peptide, thus forming a nanoparticle-hydrogel composite. The hydrogel could be delivered by syringe or spray to surfaces, mold to contours, adhere and maintain integrity in-vitro as well as in-vivo. The composite material was nontoxic to MPM cells and demonstrated the efficient release of miRNA-peptide nanoparticles from the large hydrogel matrix in-vitro. Tracking of Cy3-labeled scrambled miRNA-nanoparticles by confocal microscopy showed nanoparticle uptake by cells was accomplished via a clathrin-dependent endocytosis mechanism. Furthermore, we determined that late endosome escape permitted the release of intact miRNA into the cytoplasm of MPM cells. Pathologic analysis showed macrophages degrading the hydrogel material in-vivo. Cy3-miRNA-hydrogel composite injected subcutaneously in mice showed sustained miRNA release over three weeks. In mice bearing luciferase-positive MPM xenografts (pleural or peritoneal) treated locally with Cy3-miRNA-hydrogel composite, the Cy3-miRNA was concentrated in tumor cells but not in other organs. miRNAs targeting specific molecular vulnerabilities of MPM were used to study the therapeutic efficacy of miRNA-nanoparticle-hydrogel composites. Subcutaneous xenografts treated locally with MPM-specific miRNA-hydrogel composite shrank significantly. Similarly, pleural and peritoneal orthotopic xenografts treated with specific miRNA-hydrogel composite regressed over time. Kaplan-Meier analysis showed that miRNA-hydrogel treatment improved overall survival of tumor-bearing mice. Thus, delivery of specific miRNA via a locoregional therapeutic platform in preclinical models of MPM resulted in beneficial anti-cancer effects. This peptide-based material is promising for translational studies of nucleic acid-based locoregional therapy against MPM. Citation Format: Anand Singh, Poulami Majumder, Joel Schneider, Chuong Dinh Hoang. A novel peptide based microRNA nanoparticle hydrogel composite attenuates mesothelioma growth [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 1110.