Significant Reduction In Tumor Volume Research Articles

Tumor Volume Regression of Rectal Cancer in Daily MRI during Preoperative Chemoradiotherapy with Capecitabine

<h3>Purpose/Objective(s)</h3> Although cone beam computed tomography (CBCT) has been available for over a decade, the pattern of tumor volume change in rectal cancer during preoperative concurrent chemoradiotherapy (CCRT) has been under the veil due to poor soft tissue contrast of registration CBCT. This study was conducted to investigate daily rectal tumor volume change using registration magnetic resonance imaging (MRI). <h3>Materials/Methods</h3> Patients diagnosed with cT3-4 and/or cN+ rectal adenocarcinoma undergoing preoperative CCRT with capecitabine on the pelvis up to 50 Gy in 25 daily fractions from November 2018 to June 2019 were consecutively included. Rectal tumor volume was uniformly measured by single physician (YKK) in daily 0.35T MRI obtained with MR-guided linear accelerator. The mean +/- standard deviation (SD) of daily tumor volume (cc), difference of tumor volume between first fraction and daily fraction (baseline tumor volume at first fraction – daily tumor volume), and tumor volume reduction rate (%, [baseline tumor volume at first fraction – daily tumor volume]/baseline tumor volume at first fraction x 100) were calculated. Statistical significance of differences were tested using the Wilcoxon's signed rank test and paired t-test. <h3>Results</h3> Twenty patients were included. Median age was 64 and majority of the patients were male (90%). Most tumors were T3 (70%) and N1-2 (95%). Tumors were located median 6.9 cm (range: 1 – 12) above anal verge. Median follow-up was 32 months (range: 2.5 – 36.8) and 4 patients had recurred at the time of analysis (2 local and 2 distant). Tumor volume steadily regressed with daily administration of CCRT (mean 2.47 +/- 4.46 cc; Figure 1). Significant tumor volume reduction compared to baseline was observed from 4^th fraction (mean 13.95 +/- 17.78 cc; <i>P</i> < 0.0001). The difference of tumor volume between baseline and the last fraction was mean 46.62 +/- 56.01 cc (<i>P</i> < 0.0001). The tumor volume reduction rate was significantly increased since 4^th fraction (mean 16.64 +/- 10.9 %; <i>P</i> < 0.0001). The tumor volume reduction rate at the last fraction was – 68.18 +/- 15.68 % (<i>P</i> < 0.0001). <h3>Conclusion</h3> For the first time, this study demonstrated daily tumor volume regression in preoperative rectal CCRT with capecitabine using daily MRI. Steady pattern of tumor regression may be explained in part by daily administration of capecitabine. Based on the hypothesis-generating observation, this study may warrant initiation of further investigations on rectal tumor volume change with CCRT using daily MRI.

Phragmanthera austroarabica A.G.Mill. and J.A.Nyberg Triggers Apoptosis in MDA-MB-231 Cells In Vitro and In Vivo Assays: Simultaneous Determination of Selected Constituents.

Phragmanthera austroarabica (Loranthaceae), a semi-parasitic plant, is well known for its high content of polyphenols that are responsible for its antioxidant and anti-inflammatory activities. Gallic acid, catechin, and methyl gallate are bioactive metabolites of common occurrence in the family of Loranthaceae. Herein, the concentrations of these bioactive metabolites were assessed using high-performance thin layer chromatography (HPTLC). Methyl gallate, catechin, and gallic acid were scanned at 280 nm. Their concentrations were assessed as 14.5, 6.5 and 43.6 mg/g of plant dry extract, respectively. Phragmanthera austroarabica extract as well as the three pure compounds were evaluated regarding the cytotoxic activity. The plant extract exhibited promising cytotoxic activity against MDA-MB-231 breast cells with the IC50 value of 19.8 μg/mL while the tested pure compounds displayed IC50 values in the range of 21.26–29.6 μg/mL. For apoptosis investigation, P. austroarabica induced apoptotic cell death by 111-fold change and necrosis by 9.31-fold change. It also activated the proapoptotic genes markers and inhibited the antiapoptotic gene, validating the apoptosis mechanism. Moreover, in vivo studies revealed a significant reduction in the breast tumor volume and weight in solid Ehrlich carcinoma (SEC) mice. The treatment of SEC mice with P. austroarabica extract improved both hematological and biochemical parameters with amelioration in the liver and kidney histopathology to near normal. Taken together, P. austroarabica extract exhibited promising anti-cancer activity through an apoptosis-induction.

Thymoquinone induces apoptosis in temozolomide-resistant glioblastoma cells via the p38 mitogen-activated protein kinase signaling pathway.

Temozolomide (TMZ) can cross the blood-brain barrier (BBB) and deliver methyl groups to the purine (guanine) bases of DNA, leading to mispairing during DNA replication and subsequent cell death. However, increased expression of the repair enzyme methyl guanine methyltransferase (MGMT), which removes methyl groups from purine bases, counteracts methylation by TMZ. We evaluated the anticancer potential of thymoquinone (TQ), a hydrophobic flavonoid that inhibits resistance and induces apoptosis in various cancer cells, both in vitro and in vivo. In vitro experiments showed that compared with the Hs683 and M059J cell lines, U251 cells were more sensitive to TMZ. Compared to U251 cells, U251R cells, a TMZ drug-resistant strain established in this study, are characterized by increased expression of phosphorylated extracellular signal-regulated kinase (p-ERK) and MGMT. TQ treatments induced apoptosis in all cell lines. The p38 mitogen-activated protein kinase signal pathway was mainly activated in U251 and U251R cells; however, p-ERK and MGMT upregulation could not suppress TQ effects. Furthermore, si-p38 pretreatment of U251R cells in TQ treatments inhibited cell apoptosis. We speculate that TQ contributed to the phosphorylation and activation of p38, but not of ERK-induced apoptosis (irrespective of TMZ resistance). In vivo, U251R-derived tumors subcutaneously inoculated in nude mice exhibited significant tumor volume reduction after TQ or TQ + TMZ cotreatments. High-performance liquid chromatography assay confirmed the presence of TQ in murine brain tissues. Our findings demonstrate that TQ can effectively cross the BBB and function alone or in combination with TMZ to treat glioblastoma.

Colon cancer therapy with calcium phosphate nanoparticles loading bioactive compounds from Euphorbia lathyris: In vitro and in vivo assay

Amorphous calcium phosphate nanoparticles (ACP NPs) exhibit excellent biocompatibility and biodegradability properties. ACP NPs were functionalized with two coumarin compounds (esculetin and euphorbetin) extracted from Euphorbia lathyris seeds (BC-ACP NPs) showing high loading capacity (0.03% and 0.34% (w/w) for esculetin and euphorbetin, respectively) and adsorption efficiency (2.6% and 33.5%, respectively). BC-ACP NPs, no toxic to human blood cells, showed a more selective cytotoxicity against colorectal cancer (CRC) cells (T-84 cells) (IC50, 71.42 µg/ml) compared to non-tumor (CCD18) cells (IC50, 420.77 µg/ml). Both, the inhibition of carbonic anhydrase and autophagic cell death appeared to be involved in their action mechanism. Interestingly, in vivo treatment with BC-ACPs NPs using two different models of CRC induction showed a significant reduction in tumor volume (62%) and a significant decrease in the number and size of polyps. A poor development of tumor vasculature and invasion of normal tissue were also observed. Moreover, treatment increased the bacterial population of Akkermansia by restoring antioxidant systems in the colonic mucosa of mice. These results show a promising pathway to design innovative and more efficient therapies against CRC based on biomimetic calcium phosphate NPs loaded with natural products.

Preclinical Therapeutic Evaluation of Lenvatinib-Eluting Microspheres for Transcatheter Arterial Chemoembolization of Hepatocellular Carcinoma.

To evaluate the preclinical in vivo therapeutic response of Lenvatinib-eluting microspheres (LEN-EM) transcatheter arterial chemoembolization (LEN-TACE) in an hepatocellular carcinoma (HCC) rat model. Magnetic resonance imaging (MRI) visible LEN-EM was fabricated with poly(lactide-co-glycolide) and iron oxide nanoparticles by a double-emulsion method. The morphology, LEN loading/release kinetics, and MRI contrast effect of LEN-EM were evaluated. For in vivo study, N1S1 HCC rats were treated with LEN-TACE (LEN: 2.4mg/kg, n = 5) using LEN-EM, systemic LEN (LEN: 0.4mg/kg, oral gavage daily for 7days, n = 5), control (intra-arterial (IA) saline infusion, n = 5), and non-tumor control (n = 3). Tumor size changes were measured for 2weeks. Histology, comparative LEN plasma concentration, hematologic markers, liver profile, and serum chemistry among the groups were measured. LEN-EM with 33µm in average size was prepared in an optimized emulsion process. LEN loading efficiency was 58.7%. LEN was continuously released for 500h. LEN-TACE showed the delivered LEN-EM surrounding tumor tissue in MRI-T2* images. The LEN-TACE group demonstrated a statistically significant larger tumor volume reduction compared to the other groups at 2weeks post-procedure. Quantification data of Terminal deoxynucleotidyl transferase dUTP nick end labeling positive cells confirmed increased cancer cell death in the LEN-TACE group compared to control groups. Additional histology, hematologic markers, and liver profiles showed minimal side effects of LEN-TACE. LEN-TACE using IA delivery of LEN-EM demonstrated an effective therapeutic efficacy in an HCC rat animal model.

New thiazole-based derivatives as EGFR/HER2 and DHFR inhibitors: Synthesis, molecular modeling simulations and anticancer activity

New series of thiazole and imidazo[2,1-b]thiazole derivatives were synthesized and tested for their in vitro anticancer activity. Compounds 27, 34, 39 and 42–44 showed the best anticancer activity against the tested cancer cell lines with high safety profile and selectivity indices, especially MCF-7 breast cancer, compared to sorafenib. As an attempt to reveal their mode of cytotoxicity, EGFR, HER2 kinase and DHFR inhibition assays were performed. Compounds 39 and 43 were the most potent dual EGFR/HER2 kinase inhibitors, with IC50 values of 0.153 (EGFR), 0.108 (HER2) and 0.122 (EGFR), 0.078 (HER2) μM, respectively. 39 and 42 were the best DHFR inhibitors showing IC50 0.291 and 0.123 μM, respectively. 39 and 43 induced their cytotoxicity via cell cycle arrest at G1/S and G1 phases, respectively, and apoptosis rather than necrosis in the MCF-7 breast cancer cell line. In vivo anti-breast cancer assay of 39 and 43 showed significant tumor volume reduction with recovered caspase-3 immunoexpression. Modeling study results proved the importance of the 5-(4-substituted phenyl)-imidazo[2,1-b]thiazole moiety and the hydrazide side chain for the anticancer activity. The most potent compounds showed good drug-likeness features and could be used as prototypes for further optimization. 39 could be an example of a multi-targeting anticancer agent that acts by inhibiting EGFR/HER2 kinase, DHFR enzymes and cellular apoptosis.

Resolvin E1 Reduces Tumor Growth in a Xenograft Model of Lung Cancer

Inflammation plays a significant role in carcinogenesis and tumor growth. The current study was designed to test the hypothesis that resolvin E1 (RvE1) and overexpression of the receptor for RvE1 (ERV1) will prevent and/or reverse tumor generation in a gain-of-function mouse model of tumor seeding with lung cancer cells. To measure the impact of enhanced resolution of inflammation on cancer pathogenesis, ERV1-overexpressing transgenic (TG) and wild-type FVB mice were given an injection of 1×10⁶ LA-P0297 cells subcutaneously and were treated with RvE1 (100 ng; intraperitoneally) or placebo. To assess the impact of RvE1 as an adjunct to chemotherapy, ERV1-TG and wild-type FVB mice were treated with cisplatin or cisplatin + RvE1. RvE1 significantly prevented tumor growth and reduced tumor size, cyclooxygenase-2, NF-κB, and proinflammatory cytokines in TG animals as compared to wild-type animals. A significant decrease in Ki-67, vascular endothelial growth factor, angiopoietin (Ang)-1, and Ang-2 was also observed in TG animals as compared to wild-type animals. Tumor-associated neutrophils and macrophages were significantly reduced by RvE1 in transgenics (P&lt;0.001). RvE1 administration with cisplatin led to a significant reduction of tumor volume and reduced cyclooxygenase-2, NF-κB, vascular endothelial growth factor-A, Ang-1, and Ang-2. These data suggest that RvE1 prevents inflammation and vascularization, reduces tumor seeding and tumor size, and, when used as an adjunct to chemotherapy, enhances tumor reduction at significantly lower doses of cisplatin.

In vitro and in vivo antitumor studies of potential anticancer agents of platinum(II) complexes of dicyclopentadiene and dithiocarbamates§.

Three platinum(II) complexes of dicyclopentadiene (DCP) and dithiocarbamates (DTCs), namely, [Pt(η4-DCP)(Me2DTC)]PF6 (1), [Pt(η4-DCP)(Et2DTC)]PF6 (2), and [Pt(η4-DCP)(Bz2DTC)]PF6 (3) [Me2DTC=dimethyldithiocarbamate, Et2DTC=diethyldithiocarbamate, and Bz2DTC=dibenzyldithiocarbamate] were prepared and characterized by elemental analysis, IR, 1H, and 13C Nuclear Magnetic Resonance spectroscopy. The spectroscopic data indicated the coordination of both DCP and DTC ligands to platinum(II). The solution chemistry of complex 1 revealed that the complexes are stable in both dimethyl sulfoxide (DMSO) and 1:1 mixture of DMSO:H2O. In vitro cytotoxicity of the complexes relative to cisplatin was tested using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, against CHL-1 (human melanoma cancer cells), MDA-MB-231 (breast cancer cells), A549 (lung cancer cells), and B16 (murine melanoma cancer cells). The antiproliferative effect of all three prepared complexes was found to be significantly higher than cisplatin. Furthermore, flow cytometric analysis of complex 1 showed that the complex induced apoptosis, oxidative stress, mitochondrial potential depolarization and cell cycle arrest in a concentration-dependent pattern in the CHL-1 cells. Confirmation of apoptosis via gene expression analysis demonstrated down-regulation of anti-apoptotic genes and up-regulation of pro-apoptotic genes in the CHL-1 cells. Wound-healing assays also lent support to the strong cytotoxicity of the complexes. In vivo studies showed a significant reduction of tumor volume at the end of the experiment. In addition, the drug did not change the weight of the mice. In conclusion, complex 1 inhibited cell proliferation in vitro and reduced tumor growth in vivo.

Carbopol thickened nanoemulsions of chemotherapeutics to treat DMBA-induced breast cancer

Breast cancer is the most frequent invasive malignancy in women globally. The goal of this study was to prepare mitoxantrone and 4-methyl umbelliferone nanoemulsions followed by hydrogel preparation viz. MTO-NE-Gel and 4-MU-NE-Gel and characterized for various parameters like visual assessment, pH, rheology, ex vivo studies, and in vivo studies in 7,12-dimethylbenz(a)anthracene-induced breast cancer in swiss albino mice. The pH, viscosity, and spreadability were found to be 5.62±0.05, 13107 cP ±658, and 5.3 g.cm/s ±0.28 for 4-MU-NE-Gel and 5.75±0.03, 13025 cP ±705, and 4.06 g.cm/s ±0.45 for MTO-NE-Gel. The percent cumulative amount of drug permeated for 4-MU suspension, MTO Solution,4-MU-NE-Gel, and MTO-NE-Gel were found to be 51.26% ±5.22, 15.54% ±3.91, 89.093% ± 6.8, and 97.242%±4.51 respectively after 8 h which was significantly higher in hydrogel formulations. The outcome of pharmacodynamic studies reveals a significant reduction in tumor weight and tumor volume for MTO-NE-Gel and 4-MU-NE-Gel treated mice as compared to MTO-Gel and 4-MU-sus-Gel. Histopathological evaluation of mammary tumors indicates improvement in the tissue anatomical features and a significant decreasing trend in levels of tumor marker CA15-3 was also observed. Based on the promising results of this investigation,,,

Abstract 3512: Immunotherapy by STING activation in a CRISPR induce mouse model of GBM decrease tumor progression by CD8 T-cells and altered vascularity

Abstract Intrinsic malignant brain tumors, such as glioblastoma (GBM) are frequently resistant to immunotherapy with few hyper-mutated glioblastomas showing response. Here we have applied a long-term treatment with STING agonist and PD-1 inhibitors alone and in combination towards eliciting enhanced host immune response to combat GBM progression. To address these questions we generated mouse models of GBM by intracranial implantation of a murine GBM stem cell line or orthotopic CRISPR induced mutation to astrocytes. Systemic administration of STING agonist crosses the blood-brain barrier and induces a type-1 IFN signature in the brain and this was further enhanced in the tumor milieu. Treatment outcomes were addressed by MRi and treated animals showed a significant reduction in tumor volume and hence improved survival in comparison to the animals in the control group. Histochemical evaluation of tumor samples from the animals that received treatment showed elevated necrosis and aberrant/leaky vasculature in comparison to the control tumor samples. The presence of cytotoxic T-cells were similar between the groups but activated PD-1 positive T-cells were increased in treated tumors. Ex-vivo analysis of isolated T-cells from tumor baring mice confirms cytotoxic T-cells targeting the GBM cells. In conclusion, application of STING agonist promotes alteration of vasculitis and priming of T-cells in GBM results in increased survival and decreased tumor progression. These results reveal that GBM can be targeted by immune therapy through systemic activation of STING/IFN pathway. Citation Format: Justin V. Joseph, Huiqiang Cai, Brian Hansen, Soren R. Paludan, Mikkel Vendelbo, Martin K. Thomsen. Immunotherapy by STING activation in a CRISPR induce mouse model of GBM decrease tumor progression by CD8 T-cells and altered vascularity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3512.

Abstract 5733: Targeting nonsense-mediated decay restores p53 function in HPV-associated head and neck cancers

Abstract HPV-positive (HPV+) head and neck squamous cell carcinoma (HNSCC) tumors typically have p53 loss due to the activity of the human papillomavirus (HPV)-encoded E6 protein and the E6-associated protein (HPVE6-AP) which mediate the degradation of wild-type (WT) p53 (p53α). The loss of p53 is thought to be a major contributor to the pathogenesis of HPV+ HNSCC, which comprise approximately 35% of all HNSCC. Currently, standard care for HPV+HNSCC includes radiation and chemotherapy. However long-term toxicity related to these treatments is a concern, and there is a need for newer therapeutic strategies. Previously, we reported that two alternatively spliced, functional truncated isoforms of p53 (p53β and p53γ, comprising of exons 1 to 9β or 9γ, respectively) are degraded by nonsense-mediated decay (NMD), a regulator of aberrant mRNA stability. Here, using HPV+HNSCC cell line models, we show that NMD inhibition rescues p53β/γ isoforms and activates p53 pathway. Furthermore, we show that p53β/γ isoforms are more stable compared to p53α in these cells, with reduced vulnerabililty to HPVE6-AP- mediated degradation, and that p53β/γ isoforms contribute to increased expression of p53 transcriptional targets p21 and PUMA following NMD inhibition. Consistent with p53 pathway activation, NMD inhibition enhanced radiosensitivity of HNSCC cells. NMD inhibition attenuated colony forming ability and disrupted cell cycle progression. To evaluate the therapeutic implications of NMD inhibition, we assessed the in vivo growth of HPV+ UMSCC47 tumors. Nude mice were injected with UMSCC47 cells either subcutaneously or orthotopically in the tongue and randomized to receive vehicle or with an NMD inhibitor. In both tumor models, we observed a significant reduction in tumor volume with NMD inhibition as compared to the vehicle-treated animals. To investigate whether NMD inhibition induced the expression of p53β/γ isoforms and activated the p53 pathway in vivo, we collected tumor tissues from animals and evaluated expression of p53 isoforms and transcriptional targets by RT-PCR. We observed increased expression of p53γ, p21, GADD45A and PUMA mRNAs in NMD inhibitor treated UMSCC47 tumors, compared to their respective vehicle treated controls. These results identify NMD inhibition as a novel therapeutic strategy for restoration of p53 function in major subgroups of p53-deficient HPV+ HNSCC tumors. Citation Format: Jayanthi Gudikote, Tina Cascone, Alissa Poteete, Piyada Sitthideatphaiboon, Sonia Patel, Yan Yang, Fahao Zhang, Lerong Li, Li Shen, Monique Nilsson, Phillip Jones, Jing Wang, Jean-Christophe Bourdon, Faye M. Johnson, John V. Heymach. Targeting nonsense-mediated decay restores p53 function in HPV-associated head and neck cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5733.

Abstract 210: Development and characterization of a novel immunocompetent mouse model of radioresistant lung cancer

Abstract Standard treatment for inoperable or unresectable stage I-III NSCLC includes stereotactic ablative radiotherapy (SABR) and concurrent chemo and radiotherapy, but locoregional recurrence occurs in 10%-30% of the patients. In the absence of distant metastasis, re-irradiation is the main curative-intent treatment option available for these patients, but the risk of severe adverse effects and tumor resistance of heavily irradiated patients are challenges to this therapy. On the other hand, the combination between re-irradiation with chemotherapy or immunotherapy is a scenario not extensively studied in the literature. Current existing models have been developed in immunodeficient mice, where the effect of immunotherapy cannot be assessed. To address this issue, we have developed and characterized a lung cancer model of radiation resistance, which can be used in syngeneic mice with fully functional immune system. To this aim, we have generated a mouse adenocarcinoma radioresistant cell line (Lacun3 RR) from previously characterized Lacun3 adenocarcinoma cells. This radioresistant cell line was generated after several rounds of radiation (8 Gy) administered to the parental Lacun3 cells in vitro. Clonogenic and apoptotic assays confirmed that Lacun3 RR were more resistant to radiotherapy than the parental cell line, within the 2-4 Gy range. In addition, in vivo assays performed by subcutaneous injection of these cells showed that Lacun3 RR were completely refractory to 10 Gy irradiation, whereas a significant reduction in tumor volume (~60%) was found for parental Lacun3-derived tumors. Phenotypic characterization of these cells showed that Lacun3 RR cells had increased properties of cancer stem cells (CSC), with significantly higher ability to form tumorspheres. Lacun3 RR cells also showed increased CSC markers (ALDH1, NOTCH1) and slightly lower proliferation rates in vitro than Lacun3 cells. Cell signaling analysis revealed an increase in phosphorylated (p)p38 MAPK and pAKT basal and post-irradiation levels, and lower pH2AX (suggesting less DNA damage) in Lacun3 RR cells. RNAseq and DNAseq (Whole Exome Sequencing) were performed in both cell lines. As expected, we found that a substantial number of genes differentially expressed were significantly related to DNA repair, cell cycle regulation and spindle formation. Mutational burden was estimated from the DNAseq, revealing a similar global tumor mutational burden for both cell lines. However, some mutations were exclusively found in Lacun3 RR cells. The tumor microenvironment and tumor immune populations are currently being investigated in this model. In conclusion, we have developed a mouse model to study radiation resistance in a context of complete immunity, which could be highly valuable to study possible synergistic effects of radiotherapy and immunotherapy of re-irradiated NSCLC patients. Citation Format: Sergio León, Diego Serrano, Esther Redín, Jennifer Barranco, Maeva Houry, Francisco Expósito, Ibón Tamayo, Luis Montuenga, Alfonso Calvo, Jose Javier Aristu. Development and characterization of a novel immunocompetent mouse model of radioresistant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 210.

Abstract LB165: Preclinical evaluation of carnosic acid (CA) and rosemary extract standardized to 40% carnosic acid (RE40CA) for the prevention of invasive breast cancer

Abstract Background: There is an unmet need for the development of safer and more effective therapies for the prevention of human invasive breast cancer, for which ductal carcinoma in situ (DCIS) is a precursor. Currently, anti-hormonal therapies are the only option in individuals previously diagnosed with hormone receptor positive breast cancer. However, these agents are associated with many side effects. Furthermore, there are currently no preventive options for hormone receptor negative breast cancers. To study DCIS progression, we developed a model referred to as MIND (Mouse-INtraDuctal) in which patient-derived DCIS cells and cell lines are injected intraductally and studied as they progress to invasive ductal carcinoma (IDC). Previously we provided strong evidence for the role of B Cell Lymphoma-9 (BCL9) as a potential driver of DCIS malignancy. We chose to target BCL9 using RE40CA and its active ingredient CA previously identified in a screen for inhibitors of β-catenin binding to BCL9. Experimental Strategy: CA and RE40CA were administered to DCIS.COM MIND animal models for four weeks by oral gavage or mixed in a powder diet formulation. Drug plasma levels were evaluated by LC-MS. Four weeks following drug administration, mammary glands were excised and evaluated for efficacy and expression of biomarkers of aggressive DCIS, nuclear β-catenin and BCL9. For efficacy studies, mammary glands containing DCIS like lesions were processed and subjected to immunofluorescence staining using anti-human CK-5, anti-SMA, Dapi, β-catenin and BCL9. Extent of DCIS in vivo growth, number of invasive lesions, tumor number and tumor volume were evaluated for each mammary gland. Results: Efficacy studies showed a significant reduction in the number of invasive lesions by administration of CA by oral gavage (CA OG:10mg/kg), CA at 200mg/kg diet (CA200), CA400 and RE40CA 500. A significant reduction in extent of DCIS in vivo growth was achieved by CA OG and RE40CA 500. A significant reduction in the number of tumors was achieved by CA OG, RE40CA OG (25mg/kg equivalent to CA 10mg/kg), RE40CA 100, RE40CA 500 and RE40CA 1000. A significant reduction in tumor volume was achieved by CA OG, RE40CA OG, RE40CA 100, RE40CA 500 and RE40CA 1000. Linear regression modelling was used to compare statistical differences among the groups and differences that reached p-value &amp;lt;0.05 were considered significant. Administration of CA or RE40CA by oral gavage was also associated with a significant loss of nuclear BCL9/β-catenin expression in DCIS epithelial cells. Dietary administration of CA and RE40CA as OG or mixed in powder diet showed an increasing trend in plasma concentrations as the dose was increased. Evaluation of HE stained sections of animal organs by histopathology showed no evidence in any tissue of necrosis, degeneration, inflammation or neoplasia. Additionally, no consistent lesions attributable to treatment were observed in any of the animals or groups. Conclusion: Administration of CA and RE40CA are safe and effective in prevention of DCIS malignancy. Future studies are aimed at testing the efficacy of CA/RE40CA using DCIS cell lines representing other DCIS subtypes (ER+/PR+ and HER2 overexpressing) as well as patient derived DCIS MIND animal models. Supported by NCI PREVENT Cancer Preclinical Drug Development Program, 75N91019D00016/75N91020F00001. Citation Format: Yan Hong, Aditi Rastogi, Aryamitra Banerjee, Brandon L. Plattner, Matthew Lindeblad, Alexander V. Lyubimov, Timothy Fields, Seema A. Khan, Altaf Mohammed, Jeremy J. Johnson, Fariba Behbod. Preclinical evaluation of carnosic acid (CA) and rosemary extract standardized to 40% carnosic acid (RE40CA) for the prevention of invasive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB165.

Abstract 3979: CRISPR/Cas9-mediated knockout of PLK4 results in antiproliferative response in human epidermoid carcinoma cells in vitro and in implanted xenografts

Abstract Non-melanoma skin cancers (NMSCs) constitute the largest number of cancers diagnosed in the United States, with an estimated 1 in 5 people affected by the age of 70. The most notable NMSCs are squamous cell carcinomas (SCCs) and basal cell carcinomas (BCCs), which are considered keratinocyte cancers due to their origin. Although the majority of NMSCs can be removed with surgery or curettage, these options are not always available if the tumor occurs in an inoperable location or becomes metastatic. While non-surgical treatment modalities are also currently available, they have not been fully effective in NMSC management. Therefore, novel mechanistic-based therapeutic strategies are needed. The polo-like kinases (PLKs) are a family of serine/threonine kinases that have been found to play multiple roles in regulating the cell cycle. PLK4 is a structurally unique member of this family, which has been shown to be essential for precise centriole duplication. However, PLK4 has been shown to be dysregulated in certain cancers. Though the role of PLK4 in NMSC is not fully understood, we previously showed that PLK4 was significantly overexpressed in NMSC cell lines at both mRNA and protein levels as compared to normal keratinocytes, and its small molecule inhibition resulted in anti-proliferative responses in NMSC cell lines (Cancer Res 2018; 78 [13 Suppl]: Abstract nr 547). Here, we further validated our previous observation by determining the effects of CRISPR/Cas9-mediated knockout (KO) of PLK4 in the A431 human SCC line, both in vitro and in vivo. Our data demonstrated significantly reduced cell growth of multiple A431 PLK4 KO clones compared to wild-type (WT) cells, as measured by RealTime-Glo MT cell viability and trypan blue exclusion assays. In addition, we found significant reduction in clonogenic survival of A431 PLK4 KO cells, as measured by colony formation assays. Using a Human Cancer Pathway Finder RT2 Profiler PCR array, we identified 18 of the 84 genes tested to be greater than 1.6-fold differentially regulated after PLK4 KO in these cells. Employing Ingenuity Pathway Analysis (IPA) software, we identified that the regulation of the epithelial-mesenchymal transition by growth factors was one of the top canonical pathways modulated by PLK4 KO. To determine the in vivo relevance of our in vitro data, we compared the tumorigenicity of the A431 WT and PLK4 KO cells in nu/nu mice. The mice (n=7 per group) were injected subcutaneously with 5 x 105 A431 WT or PLK4 KO cells and tumors were allowed to grow for further analyses. We found a significant reduction in tumor volume in A431 PLK4 KO xenografts as compared to A431 PLK4 WT xenografts. Taken together, these findings support our previous results that suggest PLK4 has pro-proliferative roles in NMSC and should be further studied as a potential novel target for skin cancer management. Citation Format: Mary A. Ndiaye, Debra R. Garvey, Gagan Chhabra, Chandra K. Singh, Nihal Ahmad. CRISPR/Cas9-mediated knockout of PLK4 results in antiproliferative response in human epidermoid carcinoma cells in vitro and in implanted xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3979.

Abstract 372: Biodistribution and tumor targeting of exosomes using mouse models

Abstract Biodistribution studies are relevant models for understanding the fundamental preclinical information of the distribution of drugs to the potential target organs, which provide insight on which on-target or off-target effects might be expected. One of our research focuses is the study of uptake and distribution of bovine milk- and colostrum-derived exosomes, functionalized exosomes and exosomes in polyethyleneimine (PEI) matrix (EPM) using near-infrared fluorescent dye in rodents. Previously, when DiR dye was loaded onto milk exosomes, biodistribution studies showed that route of administration had a significant influence on the tissue distribution with somewhat uniform biodistribution with oral gavage while predominated liver accumulation with the i.v. route. In this study, we show biodistribution of colostrum exosomes, EPM and tumor targeting by attaching tumor targeting ligand, folic acid (FA). We studied the biodistribution of these formulations using exosomes labeled with Alexa Fluor 750 (AF750) in wild-type mice and subcutaneous lung tumor-bearing mice. In various studies we tested: i) biodistribution of exosomes vs EPM, ii) effect of different administration routes such as intravenous (i.v.), oral (p.o.), subcutaneous (s.c.), intranasal (i.n.) and intramuscular (i.m.) on biodistribution, and iii) tumor targeting using FA-functionalized exosomes and EPM. Uniform tissue distribution was observed upon oral administration of exosomes while predominant hepatic accumulation was observed with i.v. administration. The i.n. route resulted in pre-dominant accumulation in lung, whereas i.m. and s.c. delivery had almost similar distribution as observed with i.v. route. The distribution of exosomes and EPM matrix was largely similar. We observed that the fluorescent signals from AF750-labeled FA-Exo and FA-EPM treatment revealed higher tumor accumulation of exosomes as compared to non-functionalized exosomes and EPM, respectively due to overexpression of folate receptors. Time-dependent distribution showed accumulation of EPM in tumors at later time point. The EPM formulations could be detected at the sites otherwise difficult to target such as brain and lymph nodes after systemic administration, thus indicating suitability of these formulations to cross physiological barriers. To validate the therapeutic potential, FA-EPM was loaded with 15 μg siKRAS and injected intravenously to orthotopic A549 lung tumor-bearing mice. Significant reduction in tumor volume (67%; p &amp;lt;0.001) and tumor weight (76%; p &amp;lt;0.001) was observed which corroborated the significant knockdown of KRAS protein (p &amp;lt;0.01). Thus, this novel approach can be used as a nano ‘platform’ for drug delivery due to its increased circulating half-life, high uptake by target cells, and ability to load a diverse range of pharmaceutical therapeutics including biologics such as siRNA. (Supported from Duggan Endowment and 3P biotechnologies, Inc.) Citation Format: Disha Nagesh Moholkar, Raghuram Kandimalla, Farrukh Aqil, Ramesh Gupta. Biodistribution and tumor targeting of exosomes using mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 372.

Abstract 2566: Pre-clinical efficacy of the Wnt pathway inhibitor RXC004 in combination with anti-cancer therapies

Abstract Background: RXC004, a potent and selective inhibitor of the Wnt pathway regulator Porcupine, is being investigated in phase 2 studies in patients with advanced cancers (NCT04907851 and NCT04907539). Wnt pathway alterations, including loss-of-function RNF43 mutations and RSPO gene fusions, result in higher levels of the Wnt receptor Frizzled (Fzd) on the cell surface increasing Wnt-ligand dependent signaling. These alterations, which have been shown to be sensitive to Porcupine inhibitors, are implicated in colorectal, gastric and pancreatic cancer. Pre-clinical in vivo data on the effects of RXC004 in combination with clinically relevant anti-cancer therapies have not previously been described in genetically-defined models of cancer. Methods: RXC004 was evaluated as a monotherapy, and in combination with clinically relevant chemotherapy regimens, in an RSPO-fusion colorectal xenograft model (SNU-1411). RXC004, dosed at 5mg/kg QD (5 days on/2 days off), was evaluated in combination with either the clinically relevant colorectal triplet chemotherapy regimen (5-fluorouracil 5-FU (25mg/kg), irinotecan (50mg/kg) and oxaliplatin (5mg/kg)) or doublet chemotherapy regimen (5-FU (25mg/kg), irinotecan (50mg/kg)). Each chemotherapy regimen was dosed once a week for 3 to 4 weeks either alone or in combination with RXC004, which was dosed from day 8 onwards at 5mg/kg QD (5 days on/2 days off). Efficacy was measured by tumor volume and survival endpoint. In addition, RXC004 was studied in vitro, as monotherapy and in combination with a PARP inhibitor in genetically selected cell lines with both RSPO-fusion and RNF43 mutation. Combination and monotherapy effects on proliferation and effects on downstream signaling were investigated. Results: RXC004 in combination with the triplet chemotherapy regimen led to significantly increased survival compared to either treatment alone (p&amp;lt;0.005) and significant tumor volume reduction measured at Day 22 (p&amp;lt;0.001). RXC004 in combination with the doublet chemotherapy also increased survival compared to either treatment alone (p&amp;lt;0.05), and the combination led to significantly decreased tumor volume measured at Day 25 (p&amp;lt;0.05). RXC004 treatment in genetically defined cell lines with RSPO fusions or RNF43 mutations led to a significant reduction in BRCA1 and BRCA2 gene expression. In in vitro proliferation studies a synergistic effect of RXC004 and a PARP inhibitor was demonstrated in genetically defined cell lines. Conclusion: These data demonstrate that RXC004 in combination with clinically relevant standard of care chemotherapy regimens can lead to potential benefit over chemotherapy alone. The RXC004 induced downregulation of DNA repair pathway genes observed in vitro could contribute to the beneficial effect seen in combination with a PARP inhibitor in vitro as well as the combination effect observed in vivo with standard of care chemotherapy agents. Citation Format: James Kelly, Simon Woodcock, Inder Bhamra, Cliff Jones, Richard Armer, Jane Robertson, Caroline Phillips. Pre-clinical efficacy of the Wnt pathway inhibitor RXC004 in combination with anti-cancer therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2566.

Enhanced drug release from a pH-responsive nanocarrier can augment colon cancer treatment by blocking PD-L1 checkpoint and consuming tumor glucose

In this study, it was aimed to exploit no chemical drug to treat cancer by starvation-immunotherapy combination. We used PD-L1 siRNA to down-regulate checkpoint protein PD-L1 on cancer cell surfaces, and IFN-γ was used as an immunomodulatory agent to activate the innate immune system. When PD-L1 siRNA, IFN-γ, and glucose oxidase (GOx) were co-delivered to the tumor microenvironment (TME) by a pH-sensitive drug delivery system (DDS), starvation-immunotherapy combinedly resulted in significant tumor volume reduction in a mouse model. The DDS was prepared by using PLGA polymer whose surface was coated with chitosan. IFN-γ, siRNA, and GOx entrapment efficiencies were 93.5 ± 3.1%, 65.1 ± 2.6%, and 66.2 ± 3.3%, respectively. Moreover, the size of the DDS was well below 200 nm, allowing easy sterilization and tumor accumulation by the enhanced permeability and retention (EPR) effect. In vitro pH-dependent release study indicated that in acidic pH, the developed DDS can show accelerated drug release. In a mouse model and in acidic TME, the DDS resulted in the best therapeutic effect due to pH-dependent drug release compared to unmodified DDS and free drug. Our findings reflect that high efficiency in colon cancer treatment is achieved by combination therapy over the studied monotherapies.

An alpha-fetoprotein-maytansine conjugate for the treatment of AFP receptor expressing tumors.

e15056 Background: The alpha fetoprotein (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 a novel maytansinoid toxin to a recombinant form of human AFP (ACT-101), we can selectively deliver the toxin to cancer and MDSC cells while sparing normal cells, thereby enabling a combination of targeted and immune activating approaches against the tumor. Methods: Four ACT-101-maytansinoid conjugates with different protein-toxin ratios and different linker chemistries were initially investigated in a mouse COLO-205 xenograft model, resulting in the selection of ACT-903 as the lead candidate for further development. A second study performed in the same tumor model compared the effects of single intravenous doses of ACT-903 (10-50 mg/kg) to control groups receiving either vehicle or the unconjugated protein, ACT-101 (25 mg/kg). Tumor growth, survival and clinical observations were assessed for 60 days post tumor implantation. Both serum ACT-101 and maytansinoid levels were measured following the single dose. Results: In the first study, a statistically significant reduction in tumor volume was seen for all four conjugates compared to vehicle control (p &lt; 0.05). For ACT-903, tumors continued shrinking even after treatment completion, becoming undetectable in 9 of 10 animals. All 10 mice in this group survived through Day 60 with no obvious signs of toxicity. In contrast, there were no survivors in the control group. In the single dose study of ACT-903, a significant reduction of tumor burden compared to vehicle was achieved by Day 14 (p &lt; 0.05) in both the 40 and 50 mg/kg dose groups. Mice in these 2 groups received a second dose 15 days after the first dose, based on observed tumor re-growth. Survival was significantly prolonged in the 50 mg/kg (p = 0.0037) and 40 mg/kg group (p &lt; 0.0001) with 7 of 10 in the 50 mg/kg group and 9 of 10 animals in the 40 mg/kg group surviving to Day 60. Similar doses of ACT-101 and ACT-903 produced comparable ACT-101 serum levels, suggesting that the pharmacokinetic profile of the conjugate is driven primarily by the protein component. Free maytansinoid levels at 4 hours were less than 0.01% of the injected dose of the conjugate, indicating that the conjugate is stable in circulation. Conclusions: The efficacy and tolerability of ACT-903 in an animal tumor model supports advancing this conjugate toward clinical use with a regimen of either once a week or once every other week administration.

The role of CXCR1/2 dual inhibitor ladarixin on tumor burden and immunotherapy response in pancreatic cancer.

e16304 Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies for which few effective pharmacological treatments are currently available. Immunotherapy has revolutionized the treatment of several solid tumors, yet the results obtained in PDAC have been disappointing. Although PDAC is generally considered an immune “cold” cancer, different PDAC subtypes have diverse immune tissue microenvironment (TME) components, including tumor-associated macrophages (TAMs), that might differentially influence immunotherapy responsiveness and patient survival. TAM population is plastic, switching rapidly from a pro- to an anti-tumoral behaviour, based on specific cytokines in TME. A role for the chemokines/CXCR axis in inducing M1 polarization has been proposed in several tumors, and, consequently, the potential advantage of combining CXCR inhibitors with immune check-point inhibitors. Here, we evaluated the effectiveness of the CXCR1/2 inhibitor ladarixin, alone or in combination with anti-PD-1, on immune tolerance against PDAC and on tumor growth. Methods: A set of preclinical models were obtained by engrafting mouse PDAC-derived cell lines into the pancreas of recipient syngeneic immune-competent mice, as well as by orthotopically transplanting patient derived PDAC tumor into human immune system reconstituted (HIR) mice (Hu-NSG-CD34+). Tumor bearing mice were randomly assigned to receive vehicles, ladarixin, and anti-PD-1 alone or in combination with ladarixin, to evaluate the effect of treatment on tumor growth and mice survival. This work is supported by AIRC and Italian Ministry of Health grants. Results: CXCR1/2 inhibition by ladarixin reverted in vitro tumor-mediated M2 polarization and migration of Bone Marrow-Derived Macrophages. Ladarixin as a single agent significantly reduced tumor burden in cancer derived graft (CDG) models with high-immunogenic potential and significantly increased the efficacy of ICI in non-immunogenic CDG models that did not respond to anti-PD-1 treatment. In a HIR mouse model bearing the immunogenic subtype of human PDAC, ladarixin showed high efficacy as a single agent and the ability to increase the antitumor effects of anti-PD-1 with a statistically significant reduction of tumor volume compared to control and to single treatments (ctr vs lad, p = 0.0077; ctr vs comb, p = 0.0098; lad vs comb: 0.002; anti-PD-1 vs comb, p = 0.036). Moreover, ladarixin both as monotherapy and in combination setting increased survival (median survival: ctr 49.5 vs lad 74.5 days, p = 0.044; ctr 49.5 vs comb 150 days, p = 0.0108; anti-PD-1 57 vs comb 150 days, p = 0.046; lad 74.5 vs comb 150 days, p = 0.047). Conclusions: Ladarixin in combination with anti-PD-1 might represent an effective approach for the treatment of PDAC, converting a protumoral into an immunopermissive microenvironment in PDAC subtypes usually refractory to immunotherapy.

SERTAD1 Sensitizes Breast Cancer Cells to Doxorubicin and Promotes Lysosomal Protein Biosynthesis.

Acquired chemoresistance of tumor cells is an unwanted consequence of cancer treatment. Overcoming chemoresistance is particularly important for efficiently improving cancer therapies. Here, using multiple lines of evidence, we report the suppressive role of SERTAD1 in apoptosis/anoikis. Among various breast cancer cell lines, higher SERTAD1 expression was found in MCF7 and MDA-MB-231 in suspension than in adherent cell culture. We revealed an unexpected phenomenon that different types of cell deaths were induced in response to different doses of doxorubicin (Dox) in breast cancer cells, presumably via lysosomal membrane permeabilization. A low dose of Dox highly activated autophagy, while a high dose of the chemotherapy induced apoptosis. Inhibition of SERTAD1 promoted the sensitivity of breast cancer cells to Dox and paclitaxel, leading to a significant reduction in tumor volumes of xenograft mice. Simultaneously targeting cancer cells with Dox and autophagy inhibition successfully induced higher apoptosis/anoikis. The novel role of SERTAD1 in maintaining cellular homeostasis has also been suggested in which lysosomal contents, including LAMP1, LAMP2, CTSB, and CTSD, were reduced in SERTAD1-deficient cells.