Significant Reduction In Tumor Volume Research Articles

Synergistic anticancer therapy via ferroptosis using modified bovine serum albumin nanoparticles loaded with sorafenib and simvastatin

Ferroptosis is a non-apoptotic cell death pathway characterized by the accumulation of lipid-peroxy radicals within the affected cells. Here, we investigate the synergistic capacity of sorafenib (SOR) and simvastatin (SIM) to trigger ferroptosis for cancer therapy. For precise in-vivo delivery, SOR + SIM was ratiometrically loaded in bovine serum albumin nanoparticles (BSA-NPs) modified with 4-carboxy phenylboronic acid (CPBA). The developed CPBA-BSA(SOR + SIM)-NPs revealed size of 175.2 ± 12.8 nm, with PDI of 0.22 ± 0.03 and Z-potential of −29.6 ± 4.8 mV. Significantly, CPBA-BSA(SOR + SIM)-NPs exhibited > 2 and > 5-fold reduction in IC50 values compared to individual SOR and SIM treatments respectively, in all tested cell lines. Moreover, CPBA-BSA(SOR + SIM)-NPs treated cells exhibited decrease in glutathione levels, increase in malonaldehyde levels and depolarization of mitochondrial membrane potential (JC-1 assay). Pharmacokinetic analysis revealed enhanced AUC0-∞ and MRT levels for SOR and SIM when administered as CPBA-BSA(SOR + SIM)-NPs compared to free drugs. Crucially, in in-vivo experiments, CPBA-BSA(SOR + SIM)-NPs led to a significant reduction in tumor volume compared to various control groups. Histological and biomarker analyses underscore their biocompatibility for clinical applications. In conclusion, this study highlights the potential of CPBA-BSA(SOR + SIM)-NPs as a promising strategy for inducing ferroptosis in cancer cells, concurrently improving drug delivery and therapeutic efficacy. This approach opens new avenues in cancer treatment.

Targeting VEGF/Neuropilin-2 as a Novel Approach to Induce Radiosensitivity in Triple Negative Breast Cancer

Triple-negative breast cancer (TNBC) is an aggressive phenotype with patients having more limited survival compared with other breast cancer subtypes. The high rate of locoregional/distant recurrence after primary management implies that additional treatment is necessary to optimize the sensitivity of TNBC tumors to therapy including radiation. Our lab studies the role of VEGF/Neuropilin-2 (NRP2) in mediating cancer stem cell properties such as self-renewal and therapy resistance. We sought to identify a new therapeutic approach that can induce radiosensitization in TNBC. Cell viability in response to radiotherapy in patient-derived organoids (PDO) and xenografts (PDX) was determined using CellTiter-Glo. The VEGF/NRP2 pathway was disrupted in human BT549 and murine 4T1 models using RNA interference and a function blocking antibody (aNRP2-10) provided by aTyr Pharma. Nitrite levels, a surrogate marker for NOS2 activity, were measured using Measure-IT Nitrite Assay Kit. H2DCFDA was used to measure intracellular reactive oxygen species (ROS). Clonogenic survival assays were used to calculate radiosensitivity enhancement ratios (rER) in TNBC cell lines. For our in vivo models, 4T1 xenografts were given antibody treatment every 48 hours starting a day prior to 10Gy irradiation. We identified a novel radioresistant population expressing high levels of NRP2 and NOS2 and observed that NOS2 expression was dependent on VEGF/NRP2 via Gli1 transcription. Interestingly, reducing NRP2 expression increased radiation-induced ROS because of reduced NOS2/NO levels. The downstream consequence of higher NOS2/NO was increased Nrf2 activation via KEAP1 S-nitrosylation; thus, inducing expression of antioxidant response elements. TNBC cells were radiosensitized by knocking down NRP2 (rER 1.19 - 1.34). We saw similar results when using aNRP2-10 vs IgG (rER 1.30 - 1.37). Ectopic expression of NOS2 and constitutively active Nrf2 rescued the radioresistance phenotype in the NRP2 knock-down cells (rER 0.73 - 0.82). Consistent with the cell line data, the clinically relevant models (PDOs and PDXs) had a synergistic reduction in cell viability in response to aNRP2-10 prior to radiotherapy. We also observed a significant reduction in tumor volume and increase in necrosis in vivo in response to VEGF/NRP2 inhibition during radiotherapy. Inhibiting VEGF/NRP2 induces radiosensitization of TNBC by decreasing NOS2/NO levels and, consequently, Nrf2-mediated antioxidant gene expression. The role of Gli1 as a transcription factor for NOS2 is a novel finding that provides an unexpected link between VEGF/NRP2 and redox homeostasis in response to radiation. Current Nrf2 inhibitors are non-specific and have high toxicity; thus, making VEGF/NRP2 inhibition more favorable. Our data provide a novel therapeutic strategy for targeting TNBC breast cancer.

CD3ζ as a novel predictive biomarker of PD-1 inhibitor resistance in melanoma

Malignant melanoma is the most lethal form of skin cancer, and its incidence rates are increasing in Europe, America, and Oceania countries. Despite immune checkpoint inhibitors, such as PD-1 inhibitors, have been shown to have significant therapeutic effects on malignant melanoma, many patients are unresponsive to these treatments, even emerged resistance. There is an urgent need to discover novel biomarkers that might distinguish resistant patients from responders. In this study, we used a series of bioinformatics analyses and experimental validation. The GSE65041 was used for differential expression analysis. Kaplan-Meier was used to assess the prognostic value. ESTIMATE, ssGSEA, EPIC, TIMER, quanTiseq and MCPcounter for estimation of immune infiltration in the tumor microenvironment. We eventually identified that CD3ζ was significantly down-regulated in IHC PD-L1(−) melanoma patients. Low level of CD3ζ expression possessed a poor prognosis. CD3ζ low expression population is significantly associated with lower immune infiltration. In vivo experiment, CD3ζ expression was significantly down-regulated in mice melanoma after intradermally injected with B16–F10R cells. Compared to their wildtype counterparts, melanoma resistant mice treated with nivolumab showed significant reductions in tumor volume and weight when adding CD3ζ. In vitro experiment, the addition of CD3ζ increased nivolumab effection on inhibiting B16–F10R cell viability. Our findings indicated that CD3ζ could be a novel predictive biomarker of PD-1 inhibitor resistance in melanoma.

Bevacizumab reduces peritumoral brain edema in lung cancer brain metastases after radiotherapy.

The aim of this study was to investigate the efficacy of bevacizumab (Bev) in reducing peritumoral brain edema (PTBE) after stereotactic radiotherapy (SRT) for lung cancer brain metastases. A retrospective analysis was conducted on 44 patients with lung cancer brain metastases (70 lesions) who were admitted to our oncology and Gamma Knife center from January 2020 to May 2022. All patients received intracranial SRT and had PTBE. Based on treatment with Bev, patients were categorized as SRT + Bev and SRT groups. Follow-up head magnetic resonance imaging was performed to calculate PTBE and tumor volume changes. The edema index (EI) was used to assess the severity of PTBE. Additionally, the extent of tumor reduction and intracranial progression-free survival (PFS) were compared between the two groups. The SRT + Bev group showed a statistically significant difference in EI values before and after radiotherapy (p = 0.0115), with lower values observed after treatment, but there was no difference in the SRT group (p = 0.4008). There was a difference in the distribution of EI grades in the SRT + Bev group (p = 0.0186), with an increased proportion of patients at grades 1-2 after radiotherapy, while there was no difference in the SRT group (p > 0.9999). Both groups demonstrated a significant reduction in tumor volume after radiotherapy (p < 0.05), but there was no difference in tumor volume changes between the two groups (p = 0.4089). There was no difference in intracranial PFS between the two groups (p = 0.1541). Bevacizumab significantly reduces the severity of PTBE after radiotherapy for lung cancer. However, its impact on tumor volume reduction and intracranial PFS does not reach statistical significance.

Abstract PO-093: Crenolanib improves PD-1 response and overall survival in immune checkpoint inhibitor resistant murine models of oral squamous cell carcinoma

Abstract Background: Present line indications for pembrolizumab are for patients with metastatic or unresectable recurrent (i.e. incurable) head and neck squamous cell carcinoma (HNSCC). However, only a minority of patients on immunotherapy will realize a durable survival benefit because &amp;gt;80% of patients with metastatic HNSCC do not respond to PD-1 blockade. Tumor microenvironment mesenchymal stem cells (MSCs) and cancer associated fibroblasts (CAFs) have been reported significantly contribute to chemotherapy and radiation resistance. Moreover, MSCs have been shown to contribute to an immunosuppressive tumor microenvironment by upregulating PD-L1 in breast cancer models. We have previously shown crenolanib improves MSC mediated cisplatin resistance in vitro through modulation of MSC-mediated activation of AKT signaling, therefore we hypothesize that targeting MSCs may be of therapeutic benefit alone and in combination with anti-PD1 immunotherapy. Methods: Oral cancer was induced in the buccal space of C56/BL6 mice with the murine oral cancer cell lines MOC1 or PD-1 resistant cell line, MOC2. When tumors reached approximately 5 × 5 mm, mice were treated with 15 mg/kg (low dose) or 30 mg/kg crenolanib (high dose) for five consecutive days over 3 weeks. Tissue were harvested for immunohistochemistry and immunofluorescence analysis and ex vivo cell cultures prepared for analysis by western immunoblotting and flow cytometry. Results: There was a significant reduction in tumor volume in mice bearing MOC1 and MOC2 tumors (p&amp;lt;0.03) treated with either low or high dose crenolanib compared to vehicle control. Overall survival was also significantly improved in mice bearing MOC1 tumors treated with high dose crenolanib compared to mice treated with vehicle control (p&amp;lt;0.04) and approached significance in MOC2 mice treated with low dose crenolanib. Tumor sections were imaged by immunofluorescence microscopy. There was a decrease in expression of PDGFR-α on MOC1 tumor cells and α-SMA on tumor microenvironment stromal cells in mice treated with crenolanib compared to vehicle control, suggesting crenolanib targets both cell types. There was a significant reduction in tumor volume (p&amp;lt;0.0001) and improved overall survival (p&amp;lt;0.0004) in mice bearing MOC2 tumors treated with combination crenolanib plus pembrolizumab compared to vehicle plus pembrolizumab. Conclusions: Preliminary in vivo data suggests crenolanib may be efficacious when used in combination with anti-PD1 immunotherapy by inhibiting the immunosuppressive effects of tumor microenvironment MSCs. Citation Format: Xiangfeng Shen, Katherine Gonzalez, Rico Castillo, Ashlyn Rickard, Yvonne Mowery, Tammara Watts. Crenolanib improves PD-1 response and overall survival in immune checkpoint inhibitor resistant murine models of oral squamous cell carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-093.

Exploring Sorafenib and Simvastatin Combination for Ferroptosis-Induced Cancer Treatment: Cytotoxicity Screening, In Vivo Efficacy, and Safety Assessment.

Ferroptosis, a pathway dependent on oxygen and iron catalysts, holds promise as a therapeutic approach for cancer treatment due to its manageable regulation, direct control, and immunogenic properties. The sensitivity of cancer cells to ferroptosis induction varies based on their metabolic, genetic, and signalling pathways, prompting the use of combination therapy. In this study, we conducted a screening of drug combinations, including sorafenib (SOR) with simvastatin (SIM), phenethyl isothiocyanate, and trigonelline, in MDA-MB-231, A549, and HeLa cells to assess their cytotoxicity. The SOR-SIM combination exhibited a synergistic effect in MDA-MB-231, A549, and HeLa cells, with calculated CI values of ~ 0.66, 0.53, and 0.59, respectively. Furthermore, co-treatment with ferrostatin-1 resulted in a concentration-dependent increase in the IC50 values. Additionally, SOR + SIM demonstrated a significant reduction in GSH levels, an increase in MDA levels, and mitochondrial membrane depolarization across all three cell lines, indicating their ferroptosis inducing potential. In-vivo studies showed a significant reduction in tumor volume by 3.53-, 2.55-, and 1.47-fold compared to control, SIM, and SOR, respectively. Toxicity assessments revealed insignificant changes in biomarker levels and no observable deformations in isolated organs, except for erythrocyte shrinkage and membrane scrambling effects caused by the SOR + SIM combination. Overall, our findings highlight the potential of the SOR + SIM combination as an effective strategy for cancer treatment, emphasizing the importance of further research in targeted drug delivery systemsto ensure itssafety.

High-precision targeting and destruction of cancer-associated PDGFR-β+ stromal fibroblasts through self-assembling, protein-only nanoparticles

The need for more effective and precision medicines for cancer has pushed the exploration of new materials appropriate for drug delivery and imaging, and alternative receptors for targeting. Among the most promising strategies, finding suitable cell surface receptors and targeting agents for cancer-associated platelet derived growth factor receptor β (PDGFR-β)+ stromal fibroblasts is highly appealing. As a neglected target, this cell type mechanically and biologically supports the growth, progression, and infiltration of solid tumors in non-small cell lung, breast, pancreatic, and colorectal cancers. We have developed a family of PDGFR-β-targeted nanoparticles based on biofabricated, self-assembling proteins, upon hierarchical and iterative selective processes starting from four initial candidates. The modular protein PDGFD-GFP-H6 is well produced in recombinant bacteria, resulting in structurally robust oligomeric particles that selectively penetrates into PDGFR-β+ stromal fibroblasts in a dose-dependent manner, by means of the PDGFR-β ligand PDGFD. Upon in vivo administration, these GFP-carrying protein nanoparticles precisely accumulate in tumor tissues and enlighten them for IVIS observation. When GFP is replaced by a microbial toxin, selective tumor tissue destruction is observed associated with a significant reduction in tumor volume growth. The presented data validate the PDGFR-β/PDGFD pair as a promising toolbox for targeted drug delivery in the tumor microenvironment and oligomeric protein nanoparticles as a powerful instrument to mediate highly selective biosafe targeting in cancer through non-cancer cells. Statement of significanceWe have developed a transversal platform for nanoparticle-based drug delivery into cancer-associated fibroblasts. This is based on the engineered modular protein PDGFD-GFP-H6 that spontaneously self-assemble and selectively penetrates into PDGFR-β+ stromal fibroblasts in a dose-dependent manner, by means of the PDGFR-β ligand PDGFD. In vivo, these protein nanoparticles accumulate in tumor and when incorporating a microbial toxin, they destroy tumor tissues with a significant reduction in tumor volume, in absence of side toxicities. The data presented here validate the PDGFR-β/PDGFD pair as a fully versatile toolbox for targeted drug delivery in the tumor microenvironment intended as a synergistic treatment.

Efficacy and safety of temozolomide in the treatment of aggressive pituitary neuroendocrine tumours in Spain.

Current guidelines recommend temozolomide as the first-line chemotherapy for aggressive pituitary neuroendocrine tumours. However, no clinical trials have been conducted to date and clinical experience is quite limited. We retrospectively analyzed 28 patients (9 women and 19 men), aged 46.6+16.9, with aggressive pituitary tumours (4 pituitary carcinomas and 24 aggressive adenomas) treated with temozolomide in 10 Spanish pituitary reference centres. Four patients had Cushing's disease, 9 prolactinomas and 15 clinically non-functioning pituitary tumours (seven silent corticotroph, three silent somatotroph, one silent lactotroph, one silent gondotroph and three null-cell tumours). Median size at diagnosis was 10.5 cm3 (IQR 4.7-22.5), with cavernous sinus invasion in 88% and no metastases. Pre-temozolomide treatment, these data were 5.2 cm3 (IQR 1.9-12.3), 89.3% and 14.3% (2 intracranial and 2 spinal metastases). All patients had undergone surgery (1-5 surgeries), 25 (89.3%) had received radiotherapy (7 of them reirradiated) and 13(46.4%) had received cabergoline. One patient interrupted temozolomide prematurely. The remaining 27 patients received a median of 13 cycles (range 3-66) of 5 days every 28 days, with a mean initial dose of 265 ± 73 mg when administered alone and of 133 ± 15 mg when co-administered with radiotherapy. Eight patients (29.6%) had a significant reduction (>30%) in tumour volume and 14 (51.9%) attained tumour stabilization. After a median follow-up of 29 months (IQR 10-55), 8 out of these 22 showed disease progression. A longer progression-free survival was found in the five patients who received concomitant radiotherapy. Seven patients (25%) died (all of them because of tumour progression or complications of treatments) at 77 months (IQR 42-136) after diagnosis and 29 months (IQR 16-55) after the first dose of temozolomide. Adverse effects occurred in 18 patients (14 mild and 4 moderate or severe). In conclusion, temozolomide is an effective medical treatment for aggressive pitNET and pituitary carcinomas but is sometimes followed by tumour progression. Co-administration with radiotherapy may increase progression-free survival.

The Treatment Outcomes of Radiotherapy and Surgical Treatment for Patients with Cavernous Sinus Hemangioma: A Meta-Analysis

Cavernous sinus hemangiomas (CSHs) are infrequent benign neoplasms. This meta-analysis was conducted with the objective of examining the prognostic outcomes of surgical interventions and radiotherapy in patients diagnosed with CSHs. A comprehensive literature search was performed across PubMed, Embase, and Web of Science databases, with traceability up to June 22, 2021. The evaluation of continuous variables was conducted by applying the Weighted Mean Difference (WMD) and 95% Confidence Interval (CI). A one-arm meta-analysis was used to scrutinize the tumor control rate (TCR), clinical improvement rate (CIR), recovery rates of abducens nerve palsy and visual disturbance, total resection rate (TRR), and the incidence rate of permanent nerve palsy post-treatment. In total, 29 articles were incorporated into the meta-analysis. Post-radiotherapy for CSHs, a significant reduction in tumor volume was observed (WMD [95% CI] = -17.16 [-21.52, -12.80] cm3). The TCR, CIR, recovery rate of abducens nerve palsy, and the recovery rate of visual disturbance were 97.1 (92.9, 99.7)%, 91.9 (82.3, 98.5)%, 95.6 (83.2, 100.0)%, and 86.3 (65.0, 99.5)%, respectively. Following surgical treatment, the TRR, mean intraoperative blood loss, recovery rate of visual disturbance, incidence rate of permanent nerve palsy, and recovery rate of abducens nerve palsy were 73.2 (57.1, 86.9)%, 971.17 (584.07, 1358.27) mL, 66.4 (32.4, 0.942)%, 16.0 (4.6, 31.1)%, and 70.6 (51.0, 87.7)%, respectively. Notably, the recovery rate of abducens nerve palsy post-radiotherapy was markedly higher than post-surgical treatment. The results of this meta-analysis underscore that radiotherapy is an effective and safe treatment modality for CSHs. Furthermore, the prognostic outcomes of radiotherapy demonstrated superiority over surgical intervention.

A phase 1 trial of umbilical cord blood–derived tumor-reactive PD-L1+ natural killer cells engineered to express soluble IL-15 (TRACK-NK) in patients with non–small-cell lung cancer (NSCLC) refractory to PD-1/PD-L1 inhibitors.

TPS2665 Background: The mechanism by which patients with PD-L1 – tumors can respond favorably to anti-PD-L1 monoclonal antibody therapy has been shown to involve the PD-L1+ NK cell in the tumor microenvironment (TME). PD-L1 is induced on the cell surface when NK cells recognize tumors, leading to enhanced NK-cell function without exhaustion. The PD-L1 + NK cell is capable of trafficking to the lung TME, recognizing and killing tumor cells as well as inducing activation of endogenous T cells via potent cytokine secretion, all without the expression of a chimeric antigen receptor (CAR). TRACK-NK cells are PD-L1+ NK cells derived from cord blood (CB) and engineered to express soluble IL-15 (sIL-15). The TRACK-NK cells express high levels of tumor reactive receptors that recognize "tumor stress" including DNAM-1, NKp30 and NKG2D. TRACK-NK cells induced significant reduction in tumor volume in mice injected with human A549 NSCLC cells compared to mock-transduced NK cells, or NK cells expressing sIL-15 (sIL-15-NK) but without ex vivo activation. Survival was also prolonged in the H460 NSCLC model compared to vehicle alone. We hypothesized that TRACK-NK cells can be safely administered, and will provide meaningful clinical benefit in patients with NSCLC. Methods: NCT05334329 is a first-in-human, phase 1 dose finding study of allogeneic, off-the shelf frozen and thawed TRACK-NK cells in patients with NSCLC progressing on treatment with PD-1/PD-L1 inhibitors. TRACK-NK cells (CB NK cells engineered with retroviral transduction to express sIL-15 and ex vivo expanded and activated to express PD-L1) are administered in four weekly intravenous infusions per cycle following lymphocyte depletion (Fludarabine/Cyclophosphamide x 3 days) in the outpatient setting. Patients not experiencing disease progression (per RECIST v1.1) following cycle 1 may receive a second cycle. Primary endpoints include: 1. Toxicity/adverse events as per NCI-CTCA version 5.0, and ASTCT Consensus Grading for Cytokine Release Syndrome (CRS) and Neurotoxicity; and 2. Magnitude/duration of TRACK-NK cell persistence in peripheral blood via droplet digital PCR. We are employing a Utility Based-Bayesian Optimal Interval design to direct optimal dose determination. Dose limiting toxicity is defined as any ≥ grade 3 non-hematologic toxicity; any grade 3 CRS that does not resolve to ≤ grade 1 within 7 days; any ≥ grade 4 CRS; any ≥ grade 2 neurotoxicity that does not resolve to grade 1 within 72 hours; any ≥ grade 3 autoimmune, hypersensitivity, and acute graft-versus-host reactions. Correlatives also include cytokines levels, quantity and activation of CD4 and CD8 cells in peripheral blood, and NK and T cell trafficking and TME changes in paired tumor biopsies using quantitative immunofluorescence and gene expression analysis. Clinical trial information: NCT05334329 .

A multi-targeted T-cell engagement of PD1-resistant colorectal cancer cells via delivery of glycopeptide conjugates.

e14571 Background: Glycan-based cancer immunotherapies are novel therapeutic strategies that use the pivotal role of aberrant glycosylation patterns in cancer progression. Targeting hybrid epitopes consisting of altered glycans and carrier peptides enhances the efficacy and specificity of the anti-tumor immune response. Here, we exploit ensembles of glycopeptide conjugates to steer a multi-targeted yet tumor-specific T-cell response, a technology we call GLAAD (glycopeptides with adjuvant and tumor-associated antigen delivery). By inducing T cells that recognize and eliminate tumor cells and promoting their antigenicity in a therapeutic setting, we show the potential of our GLAAD candidate GNU-101 to overcome the transient and heterogeneous responses frequently observed with traditional immunotherapies. Methods: Using the MC38 syngeneic mouse model of colorectal cancer (CRC) in a therapeutic setting, we tested whether GNU-101 reduces tumor burden when orally administered alone or combined with immune checkpoint inhibitors (ICI). On day 0, 20 C57/BL6J mice were injected subcutaneously with MC38 CRC cells in each flank. On day 6, mice were supplemented with 3% GNU-101 in the drinking water, while control mice received normal drinking water. On days 6, 9, 12, and 15, half of the mice in each group received an anti-PD1 antibody (aPD1), while the others received isotype control. On day 16, the mice were sacrificed. Results: In this therapeutic setting, a standalone therapy of GNU-101 successfully reduced tumor growth as demonstrated by a statistically significant reduction in both tumor volume and weight. As expected, the aPD1 therapy had only a non-significant effect on tumor growth. Remarkably, its efficacy was improved when combined with GNU-101, leading to a statistically significant reduction of &gt;90% in tumor volume and weight. The analysis of the immune cell population in the tumor microenvironment revealed the activation of CD8 T cells and a reduction of immunosuppressive signals, which likely mediates the anti-tumor efficacy of GNU-101. This was obvious by a higher abundance of CD8 T cells producing IFNγ and secreting granzyme B &amp; perforin and, conversely, lower amounts of Tim3+ CD8 T cells and regulatory T cells (FoxP3+ CD4 T cells) found in mice treated with GNU-101 vs control mice. Conclusions: We demonstrate the immunomodulatory and therapeutic potential of GNU-101 in a pre-clinical mouse study in which the growth of established tumors was significantly reduced, either as a standalone therapy or in combination with existing treatment. By specifically promoting anti-cancer CD8+ T cells while reducing immunosuppressive signals, this easy-to-administer glycopeptide has the potential to create the conditions for long-term cancer control and improve treatment outcomes in cancer that are frequently resistant to immunotherapy, such as many epithelial cancers.

Efficacy and safety study of a novel alpha-fetoprotein (AFP)-maytansine conjugate in an ovarian xenograft model.

e14570 Background: AFP receptor is an oncofetal antigen and a novel target for cancer therapeutics. It is highly expressed on the surfaces of many cancers and Myeloid Derived Suppressor Cells (MDSCs) but absent on normal tissues. By conjugating maytansine toxin to a recombinant human AFP, we selectively deliver toxin to cancer cells and MDSCs while sparing normal cells, enabling a combination of targeted and immune activating approaches. We previously reported high efficacy of AFP-maytansine conjugate in a xenograft model of colorectal cancer. Here we report the effect of this conjugate in an ovarian xenograft model and in 2 independent patient-derived 3D ovarian organoids, supporting the wider utility of this approach. Methods: Four dose regimens of AFP-maytansine conjugate were compared to control in a mouse A2780 ovarian xenograft model. The conjugate was administered IV 10 days after implantation, when tumor volumes reached ≈150 mm3. Mice were randomized into 4 treatment and 1 control group (10 mice/group, table below). Tumor growth, survival and clinical observations were assessed for 62 days post implantation. In addition, organoids were cultured from primary reprogrammed tumor cells from 2 separate patients with high-grade serous ovarian carcinoma. Conjugate, staurosporine, thapsigargin or DMSO (control) were added to the media. Cytotoxicity was assessed by staining for AnnexinV and TMRE. Results: A statistically significant dose-dependent reduction in tumor volume (p&lt;0.001) and increase in survival (p&lt;0.002) were seen in all treatment groups relative to control. All animals in Groups 1,3 and 4 survived to the end of the study and 6 of 10 mice survived in the lowest dose group. 100% of mice in Group 1, highest dose group had complete tumor regression with no regrowth seen by day 62. In the other treatment groups majority of animals had complete tumor regression with no regrowth. All control animals were dead by day 38 due to excessive tumor burden. No signs of toxicity, such as ocular toxicity or vascular scarring of the tail were seen. All mice gained weight during the study. Gross pathology at the time of sacrifice was unremarkable. In addition, in the 2 ovarian organoids the AFP-maytansine conjugate demonstrated a cytotoxic effect over 72 hours exposure similar to that of thapsigargin and staurosporine given at known effective concentrations. Conclusions: This ovarian xenograft study confirms high activity without obvious toxicity of the novel AFP-maytansine conjugate in a 2nd tumor type. The targeted cytotoxic activity is further confirmed by the effect of the conjugate in 2 different ovarian cancer organoids. These results support advancing this novel conjugate toward clinical use with a dose-dense regimen. [Table: see text]

Targeting HE4 for treatment of endometrial cancer using a novel small molecule inhibitor.

e17616 Background: HE4 expression is upregulated in endometrial cancer and has been shown to have prognostic significance in endometrial cancer patients. High-throughput screening and lead optimization led us to discover UR-238, a novel small molecule inhibitor of HE4. The purpose of this study was to investigate the in vitro and in vivo effects of UR-238 in treatment of endometrial cancer. Methods: UR-238 was identified by focused medicinal chemistry. Six endometrial cancer cell lines (ECC1, AN3CA, HEC1A, MFE296, MFE280, and KLE) were grown in cell culture and treated with 8 concentrations of UR-238 (125 nM, 250 nM, 375 nM, 500 nM, 625 nM, 750 nM, 875 nM, and 1000 nM) or DMSO (control). Cell proliferation was assessed using SRB assays at 24 and 48 hours. Effects of UR-238 on the cell cycle were assessed by flow cytometry and related cell-cycle proteins were targeted via Western blots. An ELISA was performed to evaluate UR-238 treatment effect on HE4 expression. An AN3CA xenograft mouse model was used to assess in vivo effects of UR-238. Mice were treated with vehicle control or UR-238 (5 mg/kg, IP, 3 days/week) for 20 days (N=5/group). Tumor volume and weight were evaluated over time using a repeated measures ANOVA. Results: UR-238 resulted in decreased cell proliferation in a dose-dependent manner in all 6 endometrial cancer cell lines. UR-238 increased expression of p21 and p27 and induced M/G2 cell cycle arrest. UR-238 also downregulated HE4 expression. In vivo treatment with UR-238 of AN3CA expressing xenograft mice caused a significant reduction (p=0.02) in mean tumor volume compared with vehicle control after 20 days (control mean 1068.13 mm3, SE 239.76; UR-238 mean 265.62 mm3, SE 68.82). Conclusions: UR-238 inhibits cell proliferation in endometrial cancer cell lines and exhibits antitumorigenic effects in xenograft mouse models. Future studies are warranted as UR-238 may be a promising therapeutic agent for treatment of endometrial cancer.

The mechanism of liver X receptor regulates the balance of glycoFAsynthesis and cholesterol synthesis in clear cell renal cell carcinoma

The mechanism of liver X receptor regulates the balance of glycoFAsynthesis and cholesterol synthesis in clear cell renal cell carcinoma

Ursolic Acid against Prostate and Urogenital Cancers: A Review of In Vitro and In Vivo Studies.

Prostate cancer is the second most diagnosed form of cancer in men worldwide and accounted for roughly 1.3 million cases and 359,000 deaths globally in 2018, despite all the available treatment strategies including surgery, radiotherapy, and chemotherapy. Finding novel approaches to prevent and treat prostate and other urogenital cancers effectively is of major importance. Chemicals derived from plants, such as docetaxel and paclitaxel, have been used in cancer treatment, and in recent years, research interest has focused on finding other plant-derived chemicals that can be used in the fight against cancer. Ursolic acid, found in high concentrations in cranberries, is a pentacyclic triterpenoid compound demonstrated to have anti-inflammatory, antioxidant, and anticancer properties. In the present review, we summarize the research studies examining the effects of ursolic acid and its derivatives against prostate and other urogenital cancers. Collectively, the existing data indicate that ursolic acid inhibits human prostate, renal, bladder, and testicular cancer cell proliferation and induces apoptosis. A limited number of studies have shown significant reduction in tumor volume in animals xenografted with human prostate cancer cells and treated with ursolic acid. More animal studies and human clinical studies are required to examine the potential of ursolic acid to inhibit prostate and other urogenital cancers in vivo.

Abstract LB019: DUSP4 overexpression exerts anti-tumor effects in vivo in pancreatic cancer xenografts in nu/nu mice

Abstract Pancreatic cancer (PC) remains one of the most lethal of all human malignancies, being highly aggressive and difficult to treat if diagnosed at late stages. Therefore, it is important to dissect molecular mechanisms implicated in PC pathophysiology. Dual specificity phosphatase-4 (DUSP4) is a phosphatase known to negatively regulate the mitogen-activated protein (MAP) kinase family (MAPK/ERK, SAPK/JNK, P38), which are involved in multiple cancers, including PC. Previously, using tissue microarray containing a variety of pancreatic cancers and normal pancreatic tissues, we found that DUSP4 was significantly downregulated in human PC. We also demonstrated that forced overexpression of DUSP4 resulted in anti-proliferative responses in MIA PaCa-2 human PC cells (Cancer Res 2016; 76 (14_Supplement): 3667). Here, we expanded our study to determine, i) the effects of DUSP4 overexpression on tumorigenicity in vivo, and ii) the molecular mechanisms associated with DUSP4 overexpression-mediated anti-proliferative responses. MIA PaCa-2 cells were stably transfected with DUSP4 overexpression (OE) plasmid or its empty vector (pCMV6) control, followed by subcutaneous implantation into nu/nu mice (n=8 each group). Tumor growth was measured weekly for 6 weeks followed by excision of tumors and molecular analyses. We found significant reduction in tumor volume and weight in DUSP4 OE group. Further, we found marked reduction in p-ERK1/2 and p-P38 proteins in tumors from DUSP4 OE group. To determine the global mechanisms of DUSP4, we analyzed DUSP4-overexpressing MIA PaCa-2 cell lysates using quantitative global proteomics. Overall, 1678 proteins were identified with ≥2 unique peptides. Out of these, 54 proteins were significantly modulated ≥1.6-fold. Using Ingenuity Pathway Analysis (IPA), we found the association of modulated proteins with increased apoptosis and inhibited invasive and malignant tumors. The top five modulated proteins identified in response to DUSP4 OE were ADD3, PDLIM1, COL2A1, SLC20A1 and CALB2, with the first three being upregulated and the remaining two downregulated. Based on published studies in other cancers, these data support the tumor suppressor role of DUSP4 in PC since i) the loss of ADD3 and PDLIM1 (both cytoskeleton-related proteins) have been found to promote tumor growth, ii) higher expression of COL2A1 has been associated with delayed tumor recurrence, iii) higher expression of SLC20A1 (sodium-phosphate symporter) and CALB2 (calcium-binding protein) are known to be associated with cancer progression. The specific roles of these proteins in PC are not well known, providing us novel opportunities for future investigations. Overall, our data support the tumor suppressor role of DUSP4 in PC and warrants further research to validate our findings. Citation Format: Chandra K. Singh, Mary A. Ndiaye, Gagan Chhabra, Minakshi Nihal, Nihal Ahmad. DUSP4 overexpression exerts anti-tumor effects in vivo in pancreatic cancer xenografts in nu/nu mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB019.

Prophylactic antineoplastic activity of Toxoplasma gondii RH derived antigen against ehrlich solid carcinoma with evidence of shared antigens by comparative immunoblotting

BackgroundWith cancer cases escalation, an urgent request to develop novel combating strategies arise. Pathogen-based cancer-immunotherapy is getting more consideration. Autoclaved parasitic antigens seem promising candidates, taking steadily their first steps. Our aim was to examine the prophylactic antineoplastic activity of autoclaved Toxoplasma vaccine (ATV) and to test for the shared antigen theory between Toxoplasma gondii and cancer cells.MethodsMice were immunized with ATV followed by Ehrlich solid carcinoma (ESC) inoculation. Tumor weight, volume, histopathology, and immunohistochemistry for CD8+ T cells, Treg cells and VEGF were assessed. In addition, the proposed shared antigen theory between parasites and cancer was also verified using SDS-PAGE and immunoblotting.ResultsResults revealed powerful prophylactic activity of ATV with 13.3% inhibition of ESC incidence, significant reduction in tumor weight and volume in ATV vaccinated mice. Immunologically, significantly higher CD8+T cells and lower FOXP3+ Treg cells surrounded and infiltrated ESC in ATV immunized mice with higher CD8+T/Treg cells ratio and significant antiangiogenic effect. Moreover, SDS-PAGE and immunoblotting showed four shared bands between Ehrlich carcinoma and ATV of approximate molecular weights 60, 26, 22 and 12.5 KDa.ConclusionExclusively, we demonstrated a prophylactic antineoplastic activity of autoclaved Toxoplasma vaccine against ESC. Moreover, to the best of our knowledge this is the first report highlighting the existence of cross-reactive antigens between Toxoplasma gondi parasite and cancer cells of Ehrlich carcinoma.

Abstract 3480: Modulating DNA and histone methylation as a therapeutic target for non-small cell lung cancer by shifting the composition of immune cells

Abstract Lung cancer is one of the most common malignancies worldwide with non-small cell lung cancer (NSCLC) being the dominant subtype. Surgical resection remains the best treatment for NSCLC, but late diagnosis with associated metastasis precludes this as a viable modality for most patients. Furthermore, other treatments such as targeted therapies, cytotoxics, and immunotherapy only benefit a minority of patients. Therefore, new therapeutic strategies are needed to benefit more patients presenting with this devastating disease. Recent studies reveal that DNMT inhibitor (DNMTi) promotes tumor-infiltrative cytotoxic CD8+ T cells, shifts myeloid-derived suppressor cells (MDSCs) to anti-tumor macrophage-like phenotype, and enhances immunotherapy efficacy. However, some immune-regulatory genes are silenced by H3K27me3, which is facilitated by the transcriptional repressor EZH2. Additionally, conditional ablation of Ezh2 in T cells boosts effector functions of CD8+ T cells and prevents the development of regulatory T cells. Given the above, the application of combining DNMTi and EZH2i might be a viable path to facilitate tumor immune microenvironment remodeling. In this study, we aimed to define the therapeutic potential of combining DNMT and EZH2 inhibition to facilitate an anti-tumor immune response. First, we explored the potential of combined DNMTi and EZH2i in a clinically relevant murine model of NSCLC, Lewis Lung Carcinoma 1 (LLC1). Therapeutic application in this model led to a significant reduction in tumor volume. Our transcriptomic data revealed DNMTi facilitated transcriptional perturbation of both coding and transposable element-related genes, which is driven further by EZH2i combination. Immune deconvolution analysis uncovers combination therapy-induced shifts in tumor immune-cellularity, which is most profound for myeloid-associated populations. Orthogonal validation by flow cytometry defines significant reductions of MDSCs and regulatory T cells occurring within the tumor and associated microenvironment. Given the perturbations of T and myeloid cells observed in-vivo, we sought to define the drug effects on critical cell populations contained in these lineages. These data revealed that combining DNMTi and EZH2i promotes effector CD8+ T cells with more IFN-g secretion upon activation but halts the cells from exhaustion after chronic stimulation. Moreover, co-treatment promoted maturation, cross-presentation, and antigen-specific CD8+ T cell priming in dendritic cells (DCs). These data indicate that CD8+ T cells and DCs subjected to co-treatment might facilitate more robust tumor immune responses in the tumor microenvironment (TME). In summary, our data define an effective therapeutic paradigm for the treatment of NSCLC, which potentiates significant shifts in TME resident immune populations and dendritic cell phenotypes. Citation Format: Ying-Yu Chen. Modulating DNA and histone methylation as a therapeutic target for non-small cell lung cancer by shifting the composition of immune cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3480.

Abstract 4448: Chemokine (C-X-C motif) ligand 17 promotes cutaneous squamous cell carcinoma via modulating tumor-immune evasion

Abstract Cutaneous squamous cell carcinoma (cSCC) is a keratinocyte-derived invasive and metastatic tumor of the skin. It is the second-most commonly diagnosed form of skin cancer, striking 200,000 Americans annually. Further, organ transplant patients are at significantly increased risk of cSCC compared to the general population. Excision of cSCC of the head and neck area results in significant facial disfigurement. An increased understanding of the mechanisms involved in the pathogenesis of cSCC could identify means to prevent, inhibit, and reverse this process. Chemokine (C-X-C motif) ligand 17 (CXCL17) is the latest chemokine family member, and an increased CXCL17 protein expression was observed in both mouse and human cSCC cell lines. Further, deletion of CXCL17 was associated with significant inhibition of tumor cell-intrinsic properties such as proliferation, migration, and motility. CXCL17 is identified as a potent chemoattractant for macrophages, dendritic cells and myeloid-derived suppressive cells. Accordingly, utilizing a syngeneic, tumor-cell xenograft cSCC mouse model, we evaluated the effect of CXCL17 deletion on cSCC tumor-immune evasion and elucidated the underlying mechanism. Deletion of CXCL17 was associated with a significant reduction in tumor volume compared to the wild-type counterparts. Further, CXCL17 deleted cSCC tumor cell xenografts exhibited a significant increase in CD8+, cytotoxic T cells in the tumor microenvironment, suggesting an important role of CXCL17 in mediating tumor-immune evasion. Interestingly, treatment with CXCL17 induced macrophage M2 polarization and promoted macrophage efferocytosis via modulating efferocytotic machinery proteins such as MERTK, TIM4, GAS6 and AXL. Our studies have established substantial evidence for the role of CXCL17 in modulating tumor-cell extrinsic properties to affect the progression of cSCC. Citation Format: Alok R. Khandelwal, Rema Anisha Kandula, Emily Daniel, Md Maksudul Alam, Henry Craighead, Tara Moore-Medlin, Cherie-Ann O. Nathan. Chemokine (C-X-C motif) ligand 17 promotes cutaneous squamous cell carcinoma via modulating tumor-immune evasion. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4448.

Abstract 5865: Combinatorial activity of amivantamab and pembrolizumab in head and neck squamous cell carcinoma and lung squamous cell carcinoma expressing wild-type EGFR and MET

Abstract Introduction: Unmet needs exist for immunotherapy targeting PD-1/PD-L1 in head and neck squamous cell carcinoma (HNSCC) and lung squamous cell carcinoma (LUSC) due to its suboptimal response. Amivantamab, a bispecific antibody targeting epidermal growth factor receptor (EGFR) and c-Met, has been demonstrated to induce antibody-dependent cytotoxicity and trogocytosis in tumor cells. We hypothesized that combination of amivantamab with pembrolizumab may synergistically enhance antitumor immunity. In this study, we present comprehensive immunomodulatory and synergistic antitumor efficacy of amivantamab and pembrolizumab in humanized HNSCC and LUSC mice models. Methods: EGFR and MET-expressing tumors from a HNSCC and a LUSC patient were transplanted into Hu-CD34-NSG to establish humanized patient-derived xenograft (PDX) models. Tumor-bearing PDXs were treated with vehicle, pembrolizumab (10mpk, Q5D, n=10), amivantamab (10mpk, BIW, n=10), or a combination of pembrolizumab and amivantamab (n=10). Analysis of immune modulatory responses within the tumor microenvironment (TME) using multiplexed IHC, flow cytometry, and single cell RNA sequencing was performed. Results: Combination of amivantamab and pembrolizumab showed a significant reduction of tumor volume (p&amp;lt;0.001) compared to vehicle or single treatment in both models. Additionally, significantly longer survival was observed for combination treated compared to the vehicle treated groups (p&amp;lt;0.0001). Multispectral imaging of tumor indicated that granzyme B-producing CD8+ T cells were significantly increased within the tumor in the combination group (p&amp;lt;0.01). Further analysis of T cell subsets suggested that central memory type CD8+ T cells were increased upon combination treatment. This group also demonstrated significantly higher CEA-tetramer positive CD8+ T cells in the tumor (p&amp;lt;0.01), suggesting that cytotoxic T cells recognizing tumor specific antigens enhanced antitumor immune response. Single cell RNA sequencing analysis of HNSCC showed that an EGFRhighMEThigh cluster was enriched in the TME after pembrolizumab treatment. This subcluster had elevated glycolysis and lactic acid pathway-related genes compared to EGFRlowMETlow cluster. Lactate transporter, MCT4 (SLC16A3) and LDHA genes were dramatically increased in the EGFRhighMEThigh cluster. Elevated lactic acid pathway may lead to immune evasion in the tumor, dampening the activity of pembrolizumab. Interestingly, combination treatment with amivantamab could reduce EGFRhighMEThigh cluster, and could effectively control tumor via creating favorable immune TME. Conclusion: Our study demonstrated combinatorial benefits of amivantamab and pembrolizumab by effectively remodeling TME, providing a preclinical rationale to clinically combine amivantamab and PD-1 blockade treatments. Citation Format: Sun Min Lim, Chun-Bong Synn, Seong-san Kang, DongKwon Kim, Soo-Hwan Lee, Sujeong Baek, Seung Min Yang, Yu Jin Han, Mi hyun Kim, Heekyung Han, Kwangmin Na, Young Taek Kim, Sungwoo Lee, Mi Ran Yun, Jae Hwan Kim, Youngseon Byeon, Young Seob Kim, Jii Bum Lee, Ji Yun Lee, Chang Gon Kim, Min Hee Hong, Kyoung-Ho Pyo, Joshua Curtin, Bharvin Patel, Isabelle Bergiers. Combinatorial activity of amivantamab and pembrolizumab in head and neck squamous cell carcinoma and lung squamous cell carcinoma expressing wild-type EGFR and MET [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5865.