Metastasis In Preclinical Models Research Articles

Abstract PR-09: Temporally resolved proteomics identifies nidogen-2 as a co-target in pancreatic cancer that modulates fibrosis and therapy response

Abstract Pancreatic cancer (PC) is highly lethal, with a five-year survival rate of ∼13%. PC is characterised by progressive cancer-associated fibrosis. We and others have shown that targeting cancer fibrosis can improve chemotherapy efficacy and impair metastasis in pre-clinical models. As such, we aimed to use integrated temporal proteomics to dissect the matrix signatures of pancreatic tumours from the highly-metastatic KPC (Pdx1-Cre; LSL-K-rasG12D/+; LSL-p53R172H/+) and poorly-metastatic KPflC (Pdx1-Cre; LSL-K-rasG12D/+; LSL-p53fl/+) mouse models, with an aim to identify new metastatic drivers in this deadly disease. We collected pancreatic tissue from KPflC, KPC and wildtype (WT) controls at early (∼50 days), mid (∼90 days) and late-stage disease (∼200 days), enriched them for matrix proteins using ISDoT de-cellularisation and analysed them using data independent acquisition liquid chromatography-tandem mass spectrometry (DIA LC-MS/MS). DIA LC-MS/MS revealed an increased abundance of Nidogen-2 (NID2) in KPC tumours compared to KPflC. Organotypic matrices generated with NID2 CRISPR-interference (CRISPRi) CAFs had reduced fibrosis, shown via second harmonic generation (SHG) multiphoton imaging and Picrosirius Red/birefringence analysis. Organotypic invasion assays revealed that depletion of CAF NID2 significantly impeded the 3D invasion of cancer cells. Subcutaneous and orthotopic co-seeding experiments using NID2 CRISPRi CAFs with cancer cells showed that NID2 inhibition significantly impeded tumour growth and fibrosis. Intravital (in vivo) imaging with quantum dots revealed improved vascular patency in live NID2 targeted tumours, with improved response to chemotherapy. Strikingly, in orthotopic models, mice bearing NID2 targeting had significantly reduced liver metastasis and increased survival, revealing NID2 as a new stromal co-target in this aggressive disease. Citation Format: Paul Timpson, Brooke Pereira, Shona Ritchie, katie Gordon, Thomas Cox. Temporally resolved proteomics identifies nidogen-2 as a co-target in pancreatic cancer that modulates fibrosis and therapy response [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr PR-09.

Manipulating the EphB4-ephrinB2 axis to reduce metastasis in HNSCC.

The EphB4-ephrinB2 signaling axis has been heavily implicated in metastasis across numerous cancer types. Our emerging understanding of the dichotomous roles that EphB4 and ephrinB2 play in head and neck squamous cell carcinoma (HNSCC) poses a significant challenge to rational drug design. We find that EphB4 knockdown in cancer cells enhances metastasis in preclinical HNSCC models by augmenting immunosuppressive cells like T regulatory cells (Tregs) within the tumor microenvironment. EphB4 inhibition in cancer cells also amplifies their ability to metastasize through increased expression of genes associated with epithelial mesenchymal transition and hallmark pathways of metastasis. In contrast, vascular ephrinB2 knockout coupled with radiation therapy (RT) enhances anti-tumor immunity, reduces Treg accumulation into the tumor, and decreases metastasis. Notably, targeting the EphB4-ephrinB2 signaling axis with the engineered EphB4 ligands EFNB2-Fc-His and Fc-TNYL-RAW-GS reduces local tumor growth and distant metastasis in a preclinical model of HNSCC. Our data suggest that targeted inhibition of vascular ephrinB2 while avoiding inhibition of EphB4 in cancer cells could be a promising strategy to mitigate HNSCC metastasis.

Clinical implications of Wnt pathway genetic alterations in men with advanced prostate cancer.

Aberrant Wnt signaling has been implicated in prostate cancer tumorigenesis and metastasis in preclinical models but the impact of genetic alterations in Wnt signaling genes in men with advanced prostate cancer is unknown. We utilized the Prostate Cancer Precision Medicine Multi-Institutional Collaborative Effort (PROMISE) clinical-genomic database for this retrospective analysis. Patients with activating mutations in CTNNB1 or RSPO2 or inactivating mutations in APC, RNF43, or ZNRF3 were defined as Wnt-altered, while those lacking such alterations were defined as Wnt non-altered. We compared patient characteristics and clinical outcomes as well as co-occurring genetic alterations according to Wnt alteration status. Of the 1498 patients included, 193 (12.9%) were Wnt-altered. These men had a statistically significant 2-fold increased prevalence of liver and lung metastases as compared with Wnt non-altered patients at the time of initial diagnosis, (4.66% v 2.15% ; 6.22% v 3.07%), first metastatic disease diagnosis (10.88% v 5.29%; 13.99% v 6.21%), and CRPC development (11.40% v 6.36%; 12.95% v 5.29%). Wnt alterations were associated with more co-occurring alterations in RB1 (10.4% v 6.2%), AR (38.9% vs 25.7%), SPOP (13.5% vs 4.1%), FOXA1 (6.7% vs 2.8%), and PIK3CA (10.9% vs 5.1%). We found no significant differences in overall survival or other clinical outcomes from initial diagnosis, first metastatic disease, diagnosis of CRPC, or with AR inhibition for mCRPC between the Wnt groups. Wnt-altered patients with prostate cancer have a higher prevalence of visceral metastases and are enriched in RB1, AR, SPOP, FOXA1, and PIK3CA alterations. Despite these associations, Wnt alterations were not associated with worse survival or treatment outcomes in men with advanced prostate cancer.

A second-generation eIF4A RNA helicase inhibitor exploits translational reprogramming as a vulnerability in triple-negative breast cancer

In this study, we aimed to address the current limitations of therapies for macro-metastatic triple-negative breast cancer (TNBC) and provide a therapeutic lead that overcomes the high degree of heterogeneity associated with this disease. Specifically, we focused on well-documented but clinically underexploited cancer-fueling perturbations in mRNA translation as a potential therapeutic vulnerability. We therefore developed an orally bioavailable rocaglate-based molecule, MG-002, which hinders ribosome recruitment and scanning via unscheduled and non-productive RNA clamping by the eukaryotic translation initiation factor (eIF) 4A RNA helicase. We demonstrate that MG-002 potently inhibits mRNA translation and primary TNBC tumor growth without causing overt toxicity in mice. Importantly, given that metastatic spread is a major cause of mortality in TNBC, we show that MG-002 attenuates metastasis in pre-clinical models. We report on MG-002, a rocaglate that shows superior properties relative to existing eIF4A inhibitors in pre-clinical models. Our study also paves the way for future clinical trials exploring the potential of MG-002 in TNBC and other oncological indications.

Extracellular vesicles in gastric cancer: role of exosomal lncRNA and microRNA as diagnostic and therapeutic targets.

Extracellular vesicles (EVs), including exosomes, play a crucial role in intercellular communication and have emerged as important mediators in the development and progression of gastric cancer. This review discusses the current understanding of the role of EVs, particularly exosomal lncRNA and microRNA, in gastric cancer and their potential as diagnostic and therapeutic targets. Exosomes are small membrane-bound particles secreted by both cancer cells and stromal cells within the tumor microenvironment. They contain various ncRNA and biomolecules, which can be transferred to recipient cells to promote tumor growth and metastasis. In this review, we highlighted the importance of exosomal lncRNA and microRNA in gastric cancer. Exosomal lncRNAs have been shown to regulate gene expression by interacting with transcription factors or chromatin-modifying enzymes, which regulate gene expression by binding to target mRNAs. We also discuss the potential use of exosomal lncRNAs and microRNAs as diagnostic biomarkers for gastric cancer. Exosomes can be isolated from various bodily fluids, including blood, urine, and saliva. They contain specific molecules that reflect the molecular characteristics of the tumor, making them promising candidates for non-invasive diagnostic tests. Finally, the potential of targeting exosomal lncRNAs and microRNAs as a therapeutic strategy for gastric cancer were reviewed as wee. Inhibition of specific molecules within exosomes has been shown to suppress tumor growth and metastasis in preclinical models. In conclusion, this review article provides an overview of the current understanding of the role of exosomal lncRNA and microRNA in gastric cancer. We suggest that further research into these molecules could lead to new diagnostic tools and therapeutic strategies for this deadly disease.

ZHX2 deficiency enriches hybrid MET cells through regulating E-cadherin expression

Growing evidence indicates that the epithelial to mesenchymal (E/M) hybrid state plays a key role in tumorigenesis. Importantly, a hybrid mesenchymal to epithelial transition (MET) state in which individual cells express both epithelial and mesenchymal markers was recently identified in vivo, further strengthening the bonds between the hybrid EMT state and cancer progression. However, the role and the molecular mechanisms by which the hybrid MET state is maintained in triple-negative breast cancer cells (TNBC) remain elusive. Here, we find that loss of ZHX2 expression results in the hybrid MET phenotype in mesenchymal TNBC cells. Mechanistically, through directly binding to the CDH1 promoter, depletion of ZHX2 specifically reactivates expression of CDH1 encoding E-cadherin, an epithelial marker that is crucial for maintaining epithelial phenotype. Functionally, loss of ZHX2 expression enriches the hybrid MET cells and inhibits the migration and dissemination of TNBC cells or organoids, which could be reversed by restoration of E-cadherin. Moreover, depletion of ZHX2 suppresses lung metastasis in preclinical models of TNBC. In patients with TNBC, ZHX2 expression was amplified and negatively correlated with the expression of E-cadherin. These findings suggest that loss of ZHX2 promotes the hybrid MET state to impair TNBC progression.

Abstract A071: SOX2 induced plasticity and microenvironment lead to intratumoral and tissue-specific phenotypic heterogeneity in metastatic castration-resistant prostate cancer (mCRPC)

Abstract Currently, the development of efficacious agents against metastatic castration resistance prostate cancer is hampered by the lack of preclinical models that accurately reflect disease heterogeneity and treatment response. To overcome this barrier, we have developed a preclinical metastasis model using LuCap136 (PDM136), an androgen receptor (AR) positive patient-derived xenograft (PDX) that forms metastases in bone and soft tissues 3 weeks after intracardiac injection. With a combined PTEN −/−, TP53 −/mut, RB1 −/− genotype, PDM136 represents a subgroup of difficult-to-treat mPCRC. PDM136 is organotropic for bone. Mice usually reach the end point after 6-7 weeks due to paraplegia and/or cachexia. Metastatic cells maintain the expression of nuclear AR and epithelial marker cytokeratin 8. Surgical castration significantly inhibits metastatic progression and prolonged the survival rate, however, tumor will eventually progress to castration resistance. To our knowledge, PDM136 is the first AR-positive prostate cancer metastasis model that recapitulates the disease progress of mCRPC. To define the molecular mechanisms of castration resistance in PDM136, we performed RNA expression profiling on bone and soft tissue metastasis collected from intact and castrated mice. Differential expression analysis revealed over 500 transcripts were significantly altered after castration, with SOX2 and ASCL1 among the top up-regulated genes. Gene set enrichment analysis showed decreased AR signaling and increased neuroepithelium transformation in the castrated group. To validate the RNA-seq data, we performed IHC on proteins whose genes were identified by RNAseq including SOX2, ASCL1, as well as androgen receptor (AR), glucocorticoid receptor (GR) and neuroendocrine marker SYP. Surprisingly, we found that the expression of the five markers is organ-specific upon castration. Unsupervised clustering of the IHC stained markers from the bone of castrated mice revealed that AR and GR were undetectable in majority of foci whereas substantial numbers of foci in soft tissue metastasis expressed AR and GR at moderate intensity. Furthermore, we found that AR and GR expression were mutually exclusive in many foci from soft tissue metastasis. SOX2 and ASCL1 were mainly expressed in AR negative tumors, while SYP was expressed in a subpopulation of SOX2+ positive foci. Notably, liver metastasis had the most SYP expressing foci among all organs suggesting liver microenvironment promotes neuroendocrine differentiation. In summary, our IHC results revealed inter-tumor and organ-specific cellular heterogeneity induced by castration. PDM136 is a platform to understand the mechanism of responsible for mCRPC, but also a valuable tool for anti-mCRPC drug discovery. The co-existence of multiple phenotypes indicated that combination therapies targeting multiple pathways are essential to harness this disease. Citation Format: Anson Ku, Juan Yin, Adam G. Sowalsky, Kathleen Kelly. SOX2 induced plasticity and microenvironment lead to intratumoral and tissue-specific phenotypic heterogeneity in metastatic castration-resistant prostate cancer (mCRPC) [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A071.

Golgi's Role in the Development of Possible New Therapies in Cancer.

The Golgi apparatus is an important organelle found in most eukaryotic cells. It plays a vital role in the processing and sorting of proteins, lipids and other cellular components for delivery to their appropriate destinations within the cell or for secretion outside of the cell. The Golgi complex also plays a role in the regulation of protein trafficking, secretion and post-translational modifications, which are significant in the development and progression of cancer. Abnormalities in this organelle have been observed in various types of cancer, although research into chemotherapies that target the Golgi apparatus is still in its early stages. There are a few promising approaches that are being investigated: (1) Targeting the stimulator of interferon genes protein: The STING pathway senses cytosolic DNA and activates several signaling events. It is regulated by numerous post-translational modifications and relies heavily on vesicular trafficking. Based on some observations which state that a decreased STING expression is present in some cancer cells, agonists for the STING pathway have been developed and are currently being tested in clinical trials, showing encouraging results. (2) Targeting glycosylation: Altered glycosylation, which refers to changes in the carbohydrate molecules that are attached to proteins and lipids in cells, is a common feature of cancer cells, and there are several methods that thwart this process. For example, some inhibitors of glycosylation enzymes have been shown to reduce tumor growth and metastasis in preclinical models of cancer. (3) Targeting Golgi trafficking: The Golgi apparatus is responsible for the sorting and trafficking of proteins within the cell, and disrupting this process may be a potential therapeutic approach for cancer. The unconventional protein secretion is a process that occurs in response to stress and does not require the involvement of the Golgi organelles. P53 is the most frequently altered gene in cancer, dysregulating the normal cellular response to DNA damage. The mutant p53 drives indirectly the upregulation of the Golgi reassembly-stacking protein 55kDa (GRASP55). Through the inhibition of this protein in preclinical models, the reduction of the tumoral growth and metastatic capacity have been obtained successfully. This review supports the hypothesis that the Golgi apparatus may be the target of cytostatic treatment, considering its role in the molecular mechanisms of the neoplastic cells.

Clinical implications of Wnt signaling alterations in patients (pts) with advanced prostate cancer (aPC).

229 Background: Aberrant Wnt signaling has been implicated in prostate cancer tumorigenesis, progression, and metastasis in preclinical models. While studies have identified recurrent molecular alterations in the Wnt signaling components in about 20% of aPC pts, the clinical significance of these alterations has been incompletely characterized. Methods: PROMISE is a multi-institutional, retrospective, clinical-genomic database - inclusive of aPC pts who had tissue and/or blood-based genomic testing by commercially available CLIA-certified platforms. We evaluated outcomes in pts with alterations leading to the activation of the canonical Wnt pathway, specifically activating mutations in CTNNB1 or RSPO2 or inactivating mutations in APC, RNF43, or ZNRF3 (Wnt altered), compared to those lacking such alterations (Wnt wild type). Multiple endpoints were evaluated, including the frequency of metastatic disease to different sites and co-occurring alterations. Results: 1596 pts with aPC were included with a median age of 63 years at diagnosis. Wnt pathway alterations were identified in 12.4% (198/1596). Wnt altered pts had a statistically significant increase in liver and lung metastases compared with Wnt wild type pts at diagnosis (4.5% vs 2.1%, p=0.0438; 6.1% vs 2.9%, p=0.0292), at first metastatic disease (11.6% vs 5.4%, p= 0.0015; 14.8% vs 6.6%, p<0.0001), and at diagnosis of CRPC (14.2% vs 7.9%, p=0.01436; 16.1% vs 6.8%, p=0.0003). Fewer Wnt altered pts had bone metastases at CRPC compared with wild type pts (67.7% vs 75.2%, p=0.04948) without significant difference of bone metastases at the time of diagnosis or at the time of first metastatic disease. The frequency of metastases to other sites was similar between the cohorts. More Wnt altered pts had ductal features on histology at diagnosis compared with Wnt wild type pts (4.0% v 1.6%, p=0.02415) without difference in PSA, Gleason score, TNM stage, or presence of neuroendocrine or intraductal features. Co-occurring genomic alterations that were more common in Wnt altered pts included PTEN loss/mutation (25.3% vs 18.3%, p=0.0270), RB1 loss/mutation (10.6% vs 5.8%, p=0.0079), AR mutations or gain (37.9% vs 24.0%, p< 0.0001), and SPOP mutations (14.1% vs 3.9%, p< 0.0001) as compared with Wnt wild type pts. Conclusions: Wnt pathway alterations were associated with ductal histology, an increase in visceral metastases at all time points evaluated, and an increase in co-occurring PTEN, RB1, AR, and SPOP alterations. The clinical heterogeneity of aPC and differences in co-occurring mutations between the cohorts make isolating the effect of alterations in a single pathway challenging. Analysis of overall survival outcomes is currently in process, and future multivariable analysis is planned to adjust for established clinical factors and co-occurring mutations to identify the independent contributions of Wnt alterations to clinical outcomes.

Histone deacetylase inhibitors promote breast cancer metastasis by elevating NEDD9 expression

Histone deacetylase (HDAC) is a kind of protease that modifies histone to regulate gene expression, and is usually abnormally activated in tumors. The approved pan-HDAC inhibitors have demonstrated clinical benefits for patients in some hematologic malignancies. Only limited therapeutic success in breast cancer has been observed in clinical trials. In this study, we declare that pan-HDAC inhibitors targeting NEDD9-FAK pathway exacerbate breast cancer metastasis in preclinical models, which may severely impede their clinical success. NEDD9 is not an oncogene, however, it has been demonstrated recently that there are high level or activity changes of NEDD9 in a variety of cancer, including leukemia, colon cancer, and breast cancer. Mechanistically, pan-HDAC inhibitors enhance H3K9 acetylation at the nedd9 gene promoter via inhibition of HDAC4 activity, thus increase NEDD9 expression, and then activate FAK phosphorylation. The realization that pan-HDAC inhibitors can alter the natural history of breast cancer by increasing invasion warrants clinical attention. In addition, although NEDD9 has been reported to have a hand in breast cancer metastasis, it has not received much attention, and no therapeutic strategies have been developed. Notably, we demonstrate that FAK inhibitors can reverse breast cancer metastasis induced by upregulation of NEDD9 via pan-HDAC inhibitors, which may offer a potential combination therapy for breast cancer.

MicroRNAs in cancer metastasis: biological and therapeutic implications.

Cancer metastasis is the primary cause of cancer-related deaths. The seeding of primary tumours at a secondary site is a highly inefficient process requiring substantial alterations in the genetic architecture of cancer cells. These alterations include significant changes in global gene expression patterns. MicroRNAs are small, non-protein coding RNAs which play a central role in regulating gene expression. Here, we focus on microRNA determinants of cancer metastasis and examine microRNA dysregulation in metastatic cancer cells. We dissect the metastatic process in a step-wise manner and summarise the involvement of microRNAs at each step. We also discuss the advantages and limitations of different microRNA-based strategies that have been used to target metastasis in pre-clinical models. Finally, we highlight current clinical trials that use microRNA-based therapies to target advanced or metastatic tumours.

Abstract PR010: Development of a pre-clinical metastatic model of human sarcoma to identify therapeutic targets

Abstract Targeted therapies have led to significant advances in the treatment of multiple tumor types resulting in more effective and often, less toxic therapeutic options. In sarcomas, the development of targeted therapies has been met limited success. There are more than 70 sarcoma subtypes that vary in histology, clinical course and patient demographics. Despite these clear differences, clinically, sarcomas are treated similarly with variable efficacy. Patients with localized disease are treated with surgery, radiation, and often chemotherapy. Even with this aggressive multimodality treatment, 35% of patients will develop incurable metastatic disease. This highlights the need for additional therapies targeting micrometastatic disease or cells within the primary tumor with a high propensity for metastasis. Understanding the pathways driving the formation of sarcoma metastasis would allow for the development of new therapies. Targeted treatments are limited due to the heterogeneity of the disease and the paucity of pre-clinical models that accurately reflect the human disease. We have focused on creating in vivo models of sarcoma development and metastasis that can be used as the basis of further studies and to test potential therapeutic targets. Mesenchymal stem cells (MSCs) are the presumed cell of origin for sarcomas and therefore, the starting cell for our investigations. We hypothesized that by recreating key genetic events in human MSCs, we could generate sarcomas in vivo that are reflective of the human disease. RB1 and P53, tumor suppressors that are often mutated or functionally inactive in sarcomas were first targeted using CRISPR-Cas9 technology in MSCs that have been immortalized by human telomerase (hTERT). Genes that are overexpressed or amplified in The Cancer Genome Atlas (TCGA) data were identified and a library of potential oncogenes was generated. This library was then added to RB1-/-P53+/- cells through lentiviral transduction. Targeting of key tumor suppressors and adding oncogenic drivers resulted in the formation of high-grade human sarcomas subcutaneously. We then sought to investigate the ability of these cells to metastasize. Injection of cells intramuscularly (into the thigh) in immunocompromised mice resulted in the formation of spontaneous lung metastasis without clear evidence of disease in other organs. This pattern clinically reflects that of human disease. Comparing oncogenic genes from metastatic outgrowths to primary tumors identified KLF4, DDIT3, JUN, and KRAS as being enriched in metastatic cells. This system allows for reproducible and robust genetic manipulation of tumor cells to characterize key drivers of metastatic growth. This will result in the identification and validation of new therapeutic targets to treat or prevent metastatic disease in our patients. Citation Format: Janai R. Carr-Ascher. Development of a pre-clinical metastatic model of human sarcoma to identify therapeutic targets [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr PR010.

Abstract 577: Control of solid tumors by alphav beta3 CAR T cells is accompanied by profound tumor penetration and prevention of metastasis in pre-clinical models

Abstract Purpose: Using in vitro and xenograft models we aimed to determine the efficacy of αvβ3 CAR T cells deployed against melanoma and breast cancer tumors, two malignancies previously recognized for harnessing the αvβ3 pathway for angiogenic and invasion purposes. Procedures: CAR T cells expressing an anti-αvβ3 scFv containing either a CD28 or 4-1BB co-stimulatory domain and CD3zeta were generated by retroviral transduction. In vitro cytotoxicity of αvβ3 CAR T cells was assessed by co-culture with melanoma or breast tumor cells assayed with bioluminescent- and impedance-based methods and effector cytokine production by ELISA. Xenograft studies, including melanoma (SK-MEL-28) and orthotopic breast tumors (MD-AMB-231), were carried out in NSG mice to evaluate in vivo efficacy of systemically administered αvβ3 CAR T cells. Immunohistochemistry was performed to evaluate T cell infiltration of melanoma tumors and changes within the tumor microenvironment. NSG mice harboring orthotopic breast tumors were monitored for disease progression, development of metastases using bioluminescent imaging and flow cytometry analysis of lung tissue, and overall survival. Results: αvβ3 CAR T cells exhibited robust cytotoxicity and cytokine production against several melanoma and triple-negative breast tumor cell lines. Systemic administration of αvβ3 CAR T cells potently inhibited growth of SK-MEL-28 melanoma xenografts as demonstrated by significant differences in tumor volume relative to CD19 CAR treatment. Immunohistochemical analysis of tumors at the experimental endpoint revealed striking infiltration of residual tumors by human T cells in mice administered αvβ3.28z CARs, but to a lesser extent in αvβ3.BBz CAR-treated xenografts. This T cell infiltration was accompanied by marked expression of PD-L1 that was mostly absent in tumors of CD19 CAR-treated controls, suggesting treatment-induced up-regulation of PD-L1. In orthotopic xenografts of MD-AMB-231 breast tumors, αvβ3 CAR T cells were able to control tumor growth, prevent the formation of lung metastases, and resulted in enhanced overall survival. Conclusions: These pre-clinical studies highlight a renewed potential for targeting integrins, specifically αvβ3, for the treatment of solid tumors. Data from this study suggests that αvβ3 CAR T cell therapy for melanoma may be further improved by combination therapy with checkpoint inhibition. Together with our prior work demonstrating robust efficacy of αvβ3 CAR T cells to treat glioblastoma and DIPG xenografts, these results highlight the broad applicability of utilizing CAR T cells targeting αvβ3 for treatment of multiple cancer types with reduced risk of on-target, off-tumor toxicity due to the restricted expression of αvβ3 in normal tissues. Results of these studies warrant further development of αvβ3 CAR T cells for clinical use. Citation Format: Dustin A. Cobb, Lixia Liu, Barbara Dziegielewska, Philip Mollica, Maria Lee, Daniel W. Lee. Control of solid tumors by alphav beta3 CAR T cells is accompanied by profound tumor penetration and prevention of metastasis in pre-clinical 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 577.

Targeting LRRC15 Inhibits Metastatic Dissemination of Ovarian Cancer.

Dissemination of ovarian cancer (OC) cells can lead to inoperable metastatic lesions in the bowel and omentum that cause patient death. Here we show that LRRC15, a type-I 15-leucine-rich repeat-containing membrane protein, highly overexpressed in OC bowel metastases compared to matched primary tumors and acts as a potent promoter of omental metastasis. Complementary models of OC demonstrated that LRRC15 expression leads to inhibition of anoikis-induced cell death and promotes adhesion and invasion through matrices that mimic omentum. Mechanistically, LRRC15 interacted with β1-integrin to stimulate activation of focal adhesion kinase (FAK) signaling. As a therapeutic proof of concept, targeting LRRC15 with the specific antibody-drug conjugate ABBV-085 in both early and late metastatic OC cell line xenograft models prevented metastatic dissemination, and these results were corroborated in metastatic patient-derived OC xenograft models. Furthermore, treatment of 3D-spheroid cultures of LRRC15-positive patient-derived ascites with ABBV-085 reduced cell viability. Overall, these data uncover a role for LRRC15 in promoting OC metastasis and suggest a novel and promising therapy to target OC metastases.

LCOR mediates interferon-independent tumor immunogenicity and responsiveness to immune-checkpoint blockade in triple-negative breast cancer.

Ligand-dependent corepressor (LCOR) mediates normal and malignant breast stem cell differentiation. Cancer stem cells (CSCs) generate phenotypic heterogeneity and drive therapy resistance, yet their role in immunotherapy is poorly understood. Here we show that immune-checkpoint blockade (ICB) therapy selects for LCORlow CSCs with reduced antigen processing/presentation machinery (APM) driving immune escape and ICB resistance in triple-negative breast cancer (TNBC). We unveil an unexpected function of LCOR as a master transcriptional activator of APM genes binding to IFN-stimulated response elements (ISREs) in an IFN signaling-independent manner. Through genetic modification of LCOR expression, we demonstrate its central role in modulation of tumor immunogenicity and ICB responsiveness. In TNBC, LCOR associates with ICB clinical response. Importantly, extracellular vesicle (EV) Lcor-messenger RNA therapy in combination with anti-PD-L1 overcame resistance and eradicated breast cancer metastasis in preclinical models. Collectively, these data support LCOR as a promising target for enhancement of ICB efficacy in TNBC, by boosting of tumor APM independently of IFN.

Pancreatic Ductal Adenocarcinoma Cortical Mechanics and Clinical Implications.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest cancers due to low therapeutic response rates and poor prognoses. Majority of patients present with symptoms post metastatic spread, which contributes to its overall lethality as the 4th leading cause of cancer-related deaths. Therapeutic approaches thus far target only one or two of the cancer specific hallmarks, such as high proliferation rate, apoptotic evasion, or immune evasion. Recent genomic discoveries reveal that genetic heterogeneity, early micrometastases, and an immunosuppressive tumor microenvironment contribute to the inefficacy of current standard treatments and specific molecular-targeted therapies. To effectively combat cancers like PDAC, we need an innovative approach that can simultaneously impact the multiple hallmarks driving cancer progression. Here, we present the mechanical properties generated by the cell’s cortical cytoskeleton, with a spotlight on PDAC, as an ideal therapeutic target that can concurrently attack multiple systems driving cancer. We start with an introduction to cancer cell mechanics and PDAC followed by a compilation of studies connecting the cortical cytoskeleton and mechanical properties to proliferation, metastasis, immune cell interactions, cancer cell stemness, and/or metabolism. We further elaborate on the implications of these findings in disease progression, therapeutic resistance, and clinical relapse. Manipulation of the cancer cell’s mechanical system has already been shown to prevent metastasis in preclinical models, but it has greater potential for target exploration since it is a foundational property of the cell that regulates various oncogenic behaviors.

Pre-clinical Models of Metastasis in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a hostile solid malignancy coupled with an extremely high mortality rate. Metastatic disease is already found in most patients at the time of diagnosis, resulting in a 5-year survival rate below 5%. Improved comprehension of the mechanisms leading to metastasis is pivotal for the development of new targeted therapies. A key field to be improved are modeling strategies applied in assessing cancer progression, since traditional platforms fail in recapitulating the complexity of PDAC. Consequently, there is a compelling demand for new preclinical models that mirror tumor progression incorporating the pressure of the immune system, tumor microenvironment, as well as molecular aspects of PDAC. We suggest the incorporation of 3D organoids derived from genetically engineered mouse models or patients as promising new tools capable to transform PDAC pre-clinical modeling and access new frontiers in personalized medicine.

Abstract 2862: BMP4 mediated suppression of cholesterol synthesis and breast cancer metastasis

Abstract Breast cancer affects one in seven women and kills more than 600,000 women each year. Compared to the 98.9% survival rate of patients with localized breast cancer, the five-year survival rate of patients with distant metastasis is only 28.1%. Using cDNA arrays and patient datasets, we found that a protein called bone morphogenetic protein 4 (BMP4) is downregulated in highly metastatic breast cancer cells and in high-grade breast tumors. BMP4 is a ligand in the TFGβ/BMP cytokine family and has the capacity to suppress TGFβ signaling through induction of SMAD6 and SMAD7. In several different preclinical metastasis models, we have shown that restored BMP4 expression in breast cancer cells with metastatic capacity does not alter primary tumor growth but results in significantly reduced metastasis to lung, liver and spine (p< 0.01), following primary tumor resection. MDA-MB-231HM cells with or without enforced BMP4 expression were recovered from primary mammary tumors and subjected to transcriptomic profiling by RNAseq. This analysis revealed that BMP4 suppressed multiple genes involved in the cholesterol biosynthesis pathway (p=2.56×10-7) and down-regulated cholesterol levels in the tumors (p=0.042). In a patient cohort of over 2000 predominantly luminal breast cancers, we found that stage, as measured by size and lymph node status, was not altered by statin use, however statin users had less high grade tumors and more luminal A tumors. In terms of recurrence, we found a marked risk reduction in patients with luminal B tumors (p=0.014) and particularly in Her2+ luminal B tumors (p=0.045). Lung, liver and brain metastases were most reduced in statin users. Current experiments involve testing a statin therapy, with the knowledge that statins are not effective in mice, but should inhibit cholesterol synthesis in human tumors in xenograft models. In addition, we are testing an inhibitor of the nuclear receptor RORγ that has been reported to activate cholesterol synthesis through SREBP2, a master regulator of cholesterol synthesis. Citation Format: Robin L. Anderson, Lap Hing Chi, Allan D. Burrows, Suraya Roslan, Andrew Redfern. BMP4 mediated suppression of cholesterol synthesis and breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2862.

Simultaneous blockage of contextual TGF-β by cyto-pharmaceuticals to suppress breast cancer metastasis

It remains challenging to treat tumor metastasis currently in the light of multiple cascade processes of tumor metastasis. Additionally, multiple clinical drugs for metastasis have quite limited therapeutic potential and even facilitate metastasis in preclinical models. Thus, potential metastasis targets and novel metastasis-directed drugs are urgently needed to be further developed. Herein, transforming growth factor-β (TGF-β) is verified to contribute to lung metastasis in a context-dependent manner in the 4T1 orthotopic tumor-bearing mice model, which induces epithelial-mesenchymal-transition (EMT) to promote tumor dissemination from the primary site and dampens the anti-tumor response of neutrophils to support tumor colonization at the metastatic niche. In view of neutrophils' superior tropism towards both inflammatory primary tumor and metastatic niche, SB525334, a TGF-β receptor inhibitor, is loaded into cationic liposome (SBLP) which is subsequently incorporated into neutrophils to yield the cyto-pharmaceuticals (SBLP/NE). The systemically infused SBLP/NE can simultaneously migrate into both primary and metastatic sites, then release SB525334 in response to tumor stimuli, and contextually inhibit TGF-β-mediated-EMT and phenotype reversal of infiltrated neutrophils, showing substantial metastasis suppression efficacy without causing any detectable toxicities. This project shifts the paradigm for metastasis suppression therapy by simultaneous blockage of contextual TGF-β using metastatic-cascades-targeting neutrophil cyto-pharmaceuticals.

Effect of cabazitaxel on macrophages improves CD47-targeted immunotherapy for triple-negative breast cancer

BackgroundLimited therapeutic options are available for triple-negative breast cancer (TNBC), emphasizing an urgent need for more effective treatment approaches. The development of strategies by targeting tumor-associated macrophages (TAMs) to stimulate...