Monitor Tumor Growth Research Articles

An optimized protocol for the generation and monitoring of conditional orthotopic lung cancer in the KP mouse model using an adeno-associated virus vector compatible with biosafety level 1

BackgroundThe inducible Kras/p53 lung adenocarcinoma mouse model, which faithfully recapitulates human disease, is routinely initiated by the intratracheal instillation of a virus-based Cre recombinase delivery system. Handling virus-based delivery systems requires elevated biosafety levels, e.g., biosafety level 2 (BSL-2). However, in experimental animal research facilities, following exposure to viral vectors in a BSL-2 environment, rodents may not be reclassified to BSL-1 according to standard practice, preventing access to small animal micro-computed tomography (micro-CT) scanners that are typically housed in general access areas such as BSL-1 rooms. Therefore, our goal was to adapt the protocol so that the Cre-induced KP mouse model could be handled under BSL-1 conditions during the entire procedure.ResultsThe Kras-Lox-STOP-Lox-G12D/p53 flox/flox (KP)-based lung adenocarcinoma mouse model was activated by intratracheal instillation of either an adenoviral-based or a gutless, adeno-associated viral-based Cre delivery system. Tumor growth was monitored over time by micro-CT. We have successfully substituted the virus-based Cre delivery system with a commercially available, gutless, adeno-associated, Cre-expressing vector that allows the KP mouse model to be handled and imaged in a BSL-1 facility. By optimizing the anesthesia protocol and switching to a microscope-guided vector instillation procedure, productivity was increased and procedure-related complications were significantly reduced. In addition, repeated micro-CT analysis of individual animals allowed us to monitor tumor growth longitudinally, dramatically reducing the number of animals required per experiment. Finally, we documented the evolution of tumor volume for different doses, which revealed that individual tumor nodules induced by low-titer AAV-Cre transductions can be monitored over time by micro-CT.ConclusionModifications to the anesthesia and instillation protocols increased the productivity of the original KP protocol. In addition, the switch to a gutless, adeno-associated, Cre-expressing vector allowed longitudinal monitoring of tumor growth under BSL-1 conditions, significantly reducing the number of animals required for an experiment, in line with the 3R principles.

Longitudinal evaluation of the biodistribution and cellular internalization of the bispecific CD3xTRP1 antibody in syngeneic mouse tumor models

BackgroundCD3 bispecific antibodies (CD3-bsAbs) require binding of both a tumor-associated surface antigen and CD3 for their immunotherapeutic effect. Their efficacy is, therefore, influenced by the tumor uptake and the extracellular...

Toluidine blue 1% eye drop versus optical coherence tomography for margin delimitation of ocular surface squamous neoplasia.

To compare the use of toluidine blue 1% eye drops with anterior segment optical coherence tomography (OCT) for the determination of tumour margins in patients with ocular surface squamous neoplasia (OSSN). The study was conducted from July 2020 to June 2021 at the Ocular Oncology department at the Federal University of São Paulo, Brazil. Slit-lamp photographs after toluidine blue staining and OCT of the anterior segment were taken on the same day from patients with OSSN. Photographs and OCT images were analyzed quantitatively using the software ImageJ and IMAGEnet®, respectively. The agreement between techniques was evaluated qualitatively through the Bland-Altman graph and quantitatively through intraclass correlation (ICC). A total of 21 participants (71.43% males) with a clinical diagnosis of OSSN were included in the study. The average + SD diameter along the chosen axes was 4.43 ± 2.08 mm with OCT of 4.37 ± 2.03 mm with toluidine blue, a difference not statistically significant (p = 0.2891). The Bland-Altman analysis indicated a good qualitative agreement between the methods, with all cases inserted within the limits of agreement from -0.3217 to 0.4268. The ICC quantitative analysis showed an almost perfect agreement of 99.57% (95%CI: 98.96-99.83%; p < 0.001). Our findings showed that OCT and toluidine eye drops are equivalent in determining margins for tumour measurements, which is particularly relevant in low-income settings where anterior segment OCT is not available. The use of toluidine blue 1% could be an useful alternative to quantify the size of the tumour, help to monitor tumour growth, and outline margins for surgical planning.

Inhibition of tumor intrinsic BANF1 activates antitumor immune responses via cGAS-STING and enhances the efficacy of PD-1 blockade

BackgroundBANF1 is well known as a natural opponent of cyclic GMP-AMP synthase (cGAS) activity on genomic self-DNA. However, the roles of BANF1 in tumor immunity remain unclear. Here, we investigate...

Measurement of mouse head and neck tumors by automated analysis of CBCT images

Animal experiments are often used to determine effects of drugs and other biological conditions on cancer progression, but poor accuracy and reproducibility of established tumor measurement methods make results unreliable. In orthotopic mouse models of head and neck cancer, tumor volumes approximated from caliper measurements are conventionally used to compare groups, but geometrical challenges make the procedure imprecise. To address this, we developed software to better measure these tumors by automated analysis of cone-beam computed tomography (CBCT) scans. This allows for analyses of tumor shape and growth dynamics that would otherwise be too inaccurate to provide biological insight. Monitoring tumor growth by calipers and imaging in parallel, we find that caliper measurements of small tumors are weakly correlated with actual tumor volume and highly susceptible to experimenter bias. The method presented provides a unique window to sources of error in a foundational aspect of preclinical head and neck cancer research and a valuable tool to mitigate them.

RNA reporter based CRISPR/Cas12a biosensing platform for sensitive detection of circulating tumor DNA.

CRISPR/Cas biotechnology provides an exceptional platform for biosensor development. To date, the reported CRISPR/Cas biosensing systems have shown extraordinary performance for nucleic acids, small molecules, small proteins and microorganism detection. The CRISPR/Cas12a biosensing system, as a typical example, has been well established and applied for both nucleic acids and non-nucleic acids target detection. However, all established CRISPR/Cas12a biosensing systems are based on DNA reporters, which potentially limits further application.In this study, we established an RNA reporter based CRISPR/Cas12a biosensing system. A basic biosensing system was evaluated, and the limit of detection was found to be 1 nM. Afterwards, we optimized this biosensing system using both temperature and chemical enhancers. The final optimal biosensing system (with DTT & 37°C) shows fluorescence signal increased by a factor of ~10 compared with the basic system. The optimal biosensing system was further applied for the detection of circulating tumor DNA (ctDNA), which shows over 4 orders of magnitude detection range from 1pM to 25 nM, with the limit of detection of 1pM. This RNA reporter based CRISPR/Cas12a biosensing system provides an effective platform for nucleic acids quantification.Clinical Relevance- This research provides a novel approach for ctDNA diagnostics, which is an attractive biomarker for noninvasive monitoring of tumor growth, response, and spread.

A deep-learning assisted bioluminescence tomography method to enable radiation targeting in rat glioblastoma

Objective. A novel solution is required for accurate 3D bioluminescence tomography (BLT) based glioblastoma (GBM) targeting. The provided solution should be computationally efficient to support real-time treatment planning, thus reducing the x-ray imaging dose imposed by high-resolution micro cone-beam CT. Approach. A novel deep-learning approach is developed to enable BLT-based tumor targeting and treatment planning for orthotopic rat GBM models. The proposed framework is trained and validated on a set of realistic Monte Carlo simulations. Finally, the trained deep learning model is tested on a limited set of BLI measurements of real rat GBM models. Significance. Bioluminescence imaging (BLI) is a 2D non-invasive optical imaging modality geared toward preclinical cancer research. It can be used to monitor tumor growth in small animal tumor models effectively and without radiation burden. However, the current state-of-the-art does not allow accurate radiation treatment planning using BLI, hence limiting BLI’s value in preclinical radiobiology research. Results. The proposed solution can achieve sub-millimeter targeting accuracy on the simulated dataset, with a median dice similarity coefficient (DSC) of 61%. The provided BLT-based planning volume achieves a median encapsulation of more than 97% of the tumor while keeping the median geometrical brain coverage below 4.2%. For the real BLI measurements, the proposed solution provided median geometrical tumor coverage of 95% and a median DSC of 42%. Dose planning using a dedicated small animal treatment planning system indicated good BLT-based treatment planning accuracy compared to ground-truth CT-based planning, where dose-volume metrics for the tumor fall within the limit of agreement for more than 95% of cases. Conclusion. The combination of flexibility, accuracy, and speed of the deep learning solutions make them a viable option for the BLT reconstruction problem and can provide BLT-based tumor targeting for the rat GBM models.

2,3,5,4'-Tetrahydroxystilbene (TG1), a Novel Compound Derived from 2,3,5,4'-Tetrahydroxystilbene-2-O-β-D-glucoside (THSG), Inhibits Colorectal Cancer Progression by Inducing Ferroptosis, Apoptosis, and Autophagy.

Colorectal cancer (CRC) is one of the deadliest cancers worldwide and long-term survival is not guaranteed in metastatic disease despite current multidisciplinary therapies. A new compound 2,3,5,4'-Tetrahydroxystilbene (TG1), derived from THSG (2,3,5,4'-Tetrahydroxystilbene-2-O-β-D-Glucoside), has been developed, and its anticancer ability against CRC is verified in this study. HCT116, HT-29, and DLD-1 were treated with TG1 and the IC50 was measured using a sulforhodamine B assay. A Xenograft mouse model was used to monitor tumor growth. Apoptosis and autophagy, induced by TG1 in CRC cells, were examined. RNA-sequencing analysis of CRC cells treated with TG1 was performed to discover underlying pathways and mechanisms. The results demonstrated that treatment with TG1 inhibited CRC proliferation in vitro and in vivo and induced apoptotic cell death, which was confirmed by Annexin V-FITC/PI staining and Western blotting. Additionally, TG1 treatment increased the level of autophagy in cells. RNA-sequencing and GSEA analyses revealed that TG1 was associated with MYC and the induction of ferroptosis. Furthermore, the ferroptosis inhibitor Bardoxolone abrogated the cytotoxic effect of TG1 in CRC cells, indicating that ferroptosis played a crucial role in TG1-induced cytotoxicity. These findings suggest that TG1 might be a potential and potent compound for clinical use in the treatment of CRC by inhibiting proliferation and inducing ferroptosis through the MYC pathway.

Metabolic reprogramming of clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is a malignancy that exhibits metabolic reprogramming as a result of genetic mutations. This reprogramming accommodates the energy and anabolic needs of the cancer cells, leading to changes in glucose, lipid, and bio-oxidative metabolism, and in some cases, the amino acid metabolism. Recent evidence suggests that ccRCC may be classified as a metabolic disease. The metabolic alterations provide potential targets for novel therapeutic interventions or biomarkers for monitoring tumor growth and prognosis. This literature review summarized recent discoveries of metabolic alterations in ccRCC, including changes in glucose, lipid, and amino acid metabolism. The development of metabolic drugs targeting these metabolic pathways was also discussed, such as HIF-2α inhibitors, fatty acid synthase (FAS) inhibitors, glutaminase (GLS) inhibitors, indoleamine 2,3-dioxygenase (IDO) inhibitors, and arginine depletion. Future trends in drug development are proposed, including the use of combination therapies and personalized medicine approaches. In conclusion, this review provides a comprehensive overview of the metabolic alterations in ccRCC and highlights the potential for developing new treatments for this disease.

Abstract A074: In vivo modeling of castration-resistant prostate cancer in the immunodeficient SRG OncoRat

Abstract Prostate cancer is the second most common cause of cancer-related deaths in American men. When locally targeted approaches fail, androgen deprivation therapy is administered to block androgen stimulated prostate cancer growth signals. Most tumors initially exhibit rapid and dramatic regression following androgen deprivation therapy. However, many patients treated with androgen deprivation therapy will have disease recurrence and develop castration-resistant prostate cancer (CRPC), which is highly metastatic with poor survival rates. To improve clinical options for CRPC, pre-clinical animal models that closely recapitulate the human disease course are an essential component of drug development. SRG OncoRats are highly immunocompromised Rag2/Il2rg double knockouts that lack mature B cells, T cells, and circulating NK cells. To establish an in vivo model of CRPC, we inoculated VCaP cells subcutaneously into the hind-flank of SRG OncoRats and monitored tumor growth. On Day 24 rats underwent orchiectomy and tumor growth was further monitored through Day 46. As seen with androgen deprivation therapy in humans, VCaP tumors initially regressed in the SRG OncoRats. However, VCaP tumors resumed growth 14 days after castration, faithfully modeling the progression of CRPC. After tumor regrowth, animals were enrolled into a therapeutic efficacy study that mimics the current clinical challenges in prostate cancer treatment. Citation Format: R. Grace Walton, Diane Begemann, Cynthia Dunn, Valeriya Steffey, Abigail Ross, Razoanul Haque, Fallon K. Noto. In vivo modeling of castration-resistant prostate cancer in the immunodeficient SRG OncoRat [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 A074.

Novel mechanisms of androgen receptor-centered transcriptional regulatory network in regulating CD8 +T cell exhaustion and sex bias in cancer

Abstract Sex bias in cancers arising from nonreproductive organs is known but poorly understood. We recently reported on a T cell-intrinsic role of androgen receptor (AR) in driving CD8 +T cell exhaustion which underlies the poorer tumor control in males (Kwon et al., Sci Immunol, 2022). This study aims to understand how AR regulates CD8 +tumor-infiltrating lymphocytes (TILs) by identifying its genome-wide targets. We created a CD8 +T cell-specific AR knockout (KO) mouse model by crossing E8I-cre mice with Ar-floxed mice, which we challenged with syngeneic bladder tumor MB49. We then monitored tumor growth and performed spectral flow cytometry using a T cell exhaustion panel. Further, we used Cleavage Under Targets and Tagmentation – sequencing (CUT&amp;Tag-seq) to map the entire AR targets in CD8 +TILs. Loss of AR in CD8 +T cells significantly slowed the growth of MB49 in male but not female mice. CD8 +TILs from male CD8 AR KO mice showed reduced TOX +TCF1 −terminally exhausted subset (69%; P≤ 0.05) and TOX expression (72%; P = 0.0583) compared to controls. Similarly, TOX expression decreased by 43% in AR-deleted CD8 +T cells following chronic TCR stimulation in vitro (P ≤ 0.0001). Finally, using CUT&amp;Tag-seq, we found that AR binds directly to promoters of multiple key transcriptional regulators of T cell exhaustion, including Tcf7 and Tox. Through identifying the genome-wide targets of AR in CD8 +TILs, we characterized the cell-intrinsic mechanism by which AR regulates CD8 +T cell exhaustion. Our work suggests AR is a master transcriptional regulator that negatively impacts CD8 +T cell immunity, which may contribute to poorer survival in males and supports combining androgen deprivation therapy with immune checkpoint blockage for cancer treatment. Special Institutional Funding

The Tian-Men-Dong decoction suppresses the tumour-infiltrating G-MDSCs via IL-1β-mediated signalling in lung cancer

Ethnopharmacological relevanceThe traditional Chinese medicine (TCM) Tian-Men-Dong decoction (TD) has been able to effectively treat lung cancer in China for thousands of years. TD improves the quality of life in lung cancer patients by promoting nourishment of yin and reducing dryness, clearing the lung and removing toxins. Pharmacological studies show that TD contains active antitumour ingredients, but its underlying mechanism remains unknown. Aim of the studyThis study aims at exploring potential mechanisms of TD in the treatment of lung cancer by regulating granulocytic-myeloid-derived suppressor cells (G-MDSCs). Materials and methodsAn orthotopic lung cancer mouse model was generated by intrapulmonary injection with LLC-luciferase cells in immunocompetent C57BL/6 mice or immunodeficient nude mice. TD/saline was orally administered once to the model mice daily for 4 weeks. Live imaging was conducted to monitor tumour growth. Immune profiles were detected by flow cytometry. H&E and ELISA were applied to test the cytotoxicity of the TD treatment. RT–qPCR and western blotting were performed to detect apoptosis-related proteins in G-MDSCs. A neutralizing antibody (anti-Ly6G) was utilized to exhaust the G-MDSCs via intraperitoneal injection. G-MDSCs were adoptively transferred from wild-type tumour-bearing mice. Immunofluorescence, TUNEL and Annexin V/PI staining were conducted to analyse apoptosis-related markers. A coculture assay of purified MDSCs and T cells labelled with CFSE was performed to test the immunosuppressive activity of MDSCs. The presence of TD/IL-1β/TD + IL-1β in purified G-MDSCs cocultured with the LLC system was used for ex vivo experiments to detect IL-1β-mediated apoptosis of G-MDSCs. ResultsTD prolonged the survival of immune competent C57BL/6 mice in an orthotopic lung cancer model, but did not have the same effect in immunodeficient nude mice, indicating that its antitumour properties of TD are exerted by regulating immunity. TD induced G-MDSC apoptosis via the IL-1β-mediated NF-κB signalling cascade leading to effectively weaken the immunosuppressive activity of G-MDSCs and promote CD8+ T-cell infiltration, which was supported by both the depletion and adoptive transfer of G-MDSCs assays. In addition, TD also showed minimal cytotoxicity both in vivo and in vitro. ConclusionThis study reveals for the first time that TD, a classic TCM prescription, is able to regulate G-MDSC activity and trigger its apoptosis via the IL-1β-mediated NF-κB signalling pathway, reshaping the tumour microenvironment and demonstrating antitumour effects. These findings provide a scientific foundation the clinical treatment of lung cancer with TD.

Abstract 1245: A novel, immune-competent, Myc-dependent model of rapid metastatic recurrence of pancreatic cancer after resection

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a resistant malignancy with dismal outcomes. Early diagnosis, systemic treatment, and surgical resection are interdependently essential in improving survival. However, 20-30% of patients who undergo primary tumor resection will experience a metastatic recurrence in the liver within six months of surgery, despite no substantial differences in clinical history. This “rapid recurrence” (rrPDAC) is poorly understood. These metastatic lesions have a few possible origins, including occult synchronous metastases and disseminated metachronous lesions. We hypothesize metastases from either origin are affected by systemic and microenvironmental changes due to surgical intervention. RNA-seq of human rrPDAC primary tumors identified increased expression of Myc-targets when compared to long-term non-recurrers. Here, we describe a novel mouse model of immune competent, surgically resected PDAC that models rapid recurrence. Utilizing novel cell lines derived from our lab’s inducible, p48-Cre-recombinase driven LSL-KrasG12D/+ LSL-ROSA-MYC+/+ (KMC) mouse model, we orthotopically implanted KMC tumor cells into the pancreas of immune competent mice and monitored tumor growth and liver metastases weekly for two weeks via ultrasound. Mice taken down at day 14 (pre-surgery) did not demonstrate micrometastases on serial liver sectioning, however circulating tumor cells were present and isolated from venous blood. Experimental mice were randomized into control (n=17), distal pancreatectomy (n=14), and sham laparotomy cohort (n=14) and metastases were tracked via ultrasound twice-weekly. Surgically resected and sham surgery mice developed liver metastases a median 12 days earlier than controls. Additionally, using cell lines derived from the more traditionally studied KPC mouse model of PDAC (Kras activating mutation and p53 loss of function mutation) and the surgical model described above, distal pancreatectomy (n=13) resulted in metastasis to the liver in a median of 9 days earlier than controls (n=12).In the rrPDAC KMC model, RNA-sequencing of liver parenchyma of mice one day after surgery showed significantly elevated levels of Saa1 mRNA compared to nonoperative controls. This is indicative of a systemic inflammatory response after surgery that we hypothesize is having pro-metastatic effects on the liver, which has been previously reported (1).This model will allow for investigation into rrPDAC and the role surgery plays in exacerbation of metastasis in humans. Our goal is to inform changes in pre- and/or post-operative standards of care for pancreatic resection surgery to reduce the risk of rapid liver metastasis following pancreas resection surgery.

Abstract 1105: Carbon ion radiotherapy eradicates glioblastoma via tumor immune environment reprograming

Abstract Background and purpose: Glioblastoma multiforme (GBM) is a stroma rich immune cold tumor with still dismal prognosis of despite multimodal therapy consisting of postoperative surgery and radiochemotherapy. Consequently, all randomized Phase III clinical GBM trials attempting to integrate immune checkpoint blockade (ICB) have failed to demonstrate efficacy. We have recently shown that tumor immune microenvironment (TIME) reprograming via Bintrafusp alpha (Anti-PD-L1/TGF-beta bispecific molecule) combined with conventional photon radiotherapy elicits beneficial effects and prolong survival in experimental models (Lan, Knoll, Moustafa et al. Cancer Cell 2021). Moreover, eradication of radioresistant i.e., stem cell like, invasive and hypoxic tumor subpopulations by high linear energy transfer (LET) carbon ion radiotherapy (CIRT) was shown to generate an immune permissive niche in experimental GBM models (Chiblak et al. JCI Insight 2019, Tang et al. IJRBP 2022). Therefore, we aimed to systematically study the effect of CIRT vs. conventional photon radiotherapy (RT) in two (GL261 and SB28) orhotopic syngeneic mouse GBM models. Methods: Tumor growth monitoring was conducted longitudinally by means of bioluminescence and small animal MR-imaging. Mice were irradiated in five fractions (Fx) with a total dose of 15Gy RT vs. 5, 10 and 15Gy CIRT. Results: Intriguingly, all GL261 harbouring mice were cured after 5 × 3Gy Fx CIRT with no sign of tumor recurrence up to 80 days post irradiation. In contrast, no tumor control was found after 5 × 3Gy Fx RT led despite a significant prolongation of OS. The tumor control probability 50% (TCP50) for CIRT in GL261 was estimated to be 10Gy in 5 Fx. To asses if the curative effect was related to tumor eradication or reconstitution of immune surveillance. GL261 tumor cells were implanted in the contralateral hemisphere of cured mice. Single cell and Bulk RNAseq as well as immunostaining were conducted to deconvolute TIME effects post CIRT. Conclusions: In line with mechanistic studies, lack of tumor growth indicating persistent immunity against GL261 was found in CIRT cured mice. Similar experiments are conducted in SB28 model and will be presented. Together, these encouraging data indicate potential benefits for high-LET CIRT in immune modulation of GBM in experimental models that warrants for study and optimization towards clinical translation. Citation Format: Federica Ciamarone, Sarah Meister, Jennifer Furkel, Maximilian Knoll, Christian Schwager, Jovana Bojcevski, Yannik Streibel, Christel Herold-Mende, Michael Breckwoldt, Juergen Debus, Ivana Dokic, Amir Abdollahi, Katharina Schregel. Carbon ion radiotherapy eradicates glioblastoma via tumor immune environment reprograming [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 1105.

Abstract 570: Dual acting bone defender agents inhibit prostate cancer-mediated bone destruction

Abstract Introduction: Bone metastasis is clinically observed in approximately 90% of patients with advanced prostate cancer (PCa). Metastatic bone destruction is driven by a self-reinforcing process coupling osteoclast (OC)-mediated bone degradation to PCa growth, and where cell movement acts to keep these two cooperating cell types in close proximity and to move them across the bone surface. We sought to create a new class of therapeutics that would be highly effective at inhibiting PCa-mediated bone destruction by targeting both OC function and cell motility. Methods: We synthesized new chemical entities, termed Dual Acting Bone Defender agents (DABDs), that couple the small molecule anti-motility agent, KBU2046, with bisphosphonate (bis), through a chemical linker. Assays for in vitro efficacy included: cell migration and invasion, binding to bone mineral, inhibiting osteoclast (OC)-mediated bone destruction, inhibition of Raf1 phosphorylation and inhibition of protein prenylation. In vivo assays included intra-cardiac (IC) injection of human PCa cells into mice, followed by real time monitoring of tumor growth and bone destruction by IVIS and CT imaging, respectively. Findings: We successfully created a novel route of chemical synthesis allowing us to access and probe unique chemical space, providing for single small molecules with bis functional groups linked to a KBU2046 core through a linker moiety. DABDs retain the cellular and molecular characteristics of KBU2046 and bis. Like KBU2046, they inhibit PCa cell motility and phosphorylation of Raf1 on its activation motif. They also inhibit the motility of breast and lung cancer cells. Like bis, they inhibit OC-mediated bone mineral destruction, prenylation of Ras small GTPase proteins, and chemically bind hydroxyapatite. Administered prior to IC injection, DABDs are so effective that they prolong animal survival and inhibit bone destruction in a dose-dependent manner. In mice with established bone metastasis to the femur, after IC injection, DABDs inhibit the growth of tumors. Doses 3,000 fold higher than that required for efficacy had no clinical or critical organ toxicity. Conclusions: DABDs represent a new multifunctional class of therapeutic agents that inhibit PCa-mediated bone destruction and have a high potential for advancing clinical efficacy in PCa patients with bone metastasis. Citation Format: Fangfang Qiao, Ryan Gordon, Abhinandan Pattanayak, Katelyn O’Neill, Nina Chaika, Weining Chen, Rachel Rhatigan, Raymond Bergan. Dual acting bone defender agents inhibit prostate cancer-mediated bone destruction [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 570.

Abstract 39: Assessment of a novel method using adult healthy donor mobilized human CD34+hematopoietic stem cells in NCG mice for use in tumor modeling

Abstract Checkpoint inhibitors including anti-PD-1/PD-L1 are a type of immunotherapy that have become the standard of care for cancer patients. Continual focus is placed on developing new treatments for cancer, including breast, lung and colon cancer, which are the most common worldwide. Humanized mice are an important in-vivo animal model that recapitulate crucial aspects of human tumor biology and anti-tumoral immunity allowing for assessment of immunotherapies. Currently these models are primarily created using cord blood, which is in limited supply. This leads to the isolation of low numbers of hCD34+ stem cells, resulting in small cohorts of animals. This limitation represents a challenge in working with humanized mice making it important to consider alternative sources of hCD34+ stem cells. In this study, we compared NCG mice engrafted with an alternative source of adult healthy donor mobilized hCD34+ stem cells to those engrafted with cord blood derived hCD34+ stem cells.After evaluating engraftment kinetics between the two cell sources we examined the anti-tumoral effect of an anti-human PD-1 checkpoint inhibitor in mice bearing three different human tumor xenograft cell lines (CDX) (A549 lung, COLO-205 colon and MDA-MB-436 breast cancer). NCG mice were humanized using either cord blood derived or adult healthy donor mobilized hCD34+ stem cells. Levels of human immune cell engraftment were measured in peripheral blood using multi-color flow cytometry. Humanized NCG mice were implanted subcutaneously with cancer cells, and tumor bearing mice (TBM) were randomized into treatment groups when the average tumor size reached comparable volumes for each tumor type. Vehicle control mice were treated with isotype control IgG antibody, whereas TBM were dosed with anti-hPD-1 antibody. Clinical observations, body weights and tumor growth kinetics were recorded throughout the study. At the time of euthanasia whole blood, spleen and tumor tissues were collected and processed for immune profiling by multi-color flow cytometry. Adult healthy donor mobilized hCD34+ humanized mice have sustained humanization levels out to 34 weeks post-injection and can be successfully engrafted with human CDX tumors. The ability to monitor tumor growth kinetics and observe a response to an anti-hPD-1 checkpoint inhibitor across multiple tumor types indicates that NCG mice humanized with adult healthy donor mobilized CD34+ stem cells are useful when assessing immunotherapies. Future directions of this model will expand beyond monotherapies to include the evaluation of additional treatment methods such as bispecific antibodies and cell-directed therapies. Citation Format: Jenny Rowe, Steven Bronson, Christoph Eberle, Ann Fiore, Bob Mihalek, Stephen Festin. Assessment of a novel method using adult healthy donor mobilized human CD34+hematopoietic stem cells in NCG mice for use in tumor modeling [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 39.

Abstract 3582: Differentiation of niraparib and olaparib brain penetration in a mouse brain metastatic tumor model

Abstract Purpose: There remains an unmet need to provide effective treatment therapy for patients with primary and metastatic brain tumors; lack of drug penetration across the blood brain barrier (BBB) is a key factor. Synthetic lethality remains an attractive mechanism in treating brain tumors post radiotherapy. Here, we evaluated brain penetration and distribution of niraparib and olaparib in a mouse brain tumor model. Experimental: Female mice (CrTac:NCr-Foxn1nu; 6 w/o) received 2.5E5 luciferase transfected human breast cancer line (MDA 231-BRM2-831) via intracardiac injection. Mice were imaged twice/week using bioluminescence imaging (BLI) to monitor tumor growth. On day 35, mice with brain metastases (BM) were treated via oral gavage once daily for 5 days with either niraparib (35 mg/kg, n=4 BM, n=3 control), olaparib (50 mg/kg, n=3 BM, n=3 control), or vehicle (n=3 control). Terminal blood samples and brains were collected 2 hr post final dose. Serial tissue sections were collected for MALDI-IMS, H&amp;E and IHC staining from 5 distinct horizontal planes in the brain. Tissue collected between each imaging plane was homogenized for LC-MS bioanalysis. Summary of Data: In vivo BLI imaging was used to identify mice with BM and tumor presence was confirmed ex vivo using IHC. Quantitative MALDI IMS of coronal brain sections collected from mice administered niraparib showed consistent concentrations distributed throughout the brain parenchyma with locally higher concentrations detected from tumor regions. Table 1 summarizes the LC-MS bioanalysis concentrations in plasma and bulk brain homogenates and brain section concentrations detected by MALDI IMS. The estimated mean unbound brain-to-plasma partition coefficient (Kp,uu,brain) was 3.0x and 5.6x higher for niraparib compared to olaparib in control and BM mice, respectively. Conclusions: Herein, we demonstrated that niraparib has a higher brain penetration and distribution compared to olaparib in both control mice and mice with BM. Summary of Quantification Results Displayed as Total Conc. with Adjusted Free Conc. in Parentheses Compound Group Terminal Plasma Mean (ng/mL) LC-MS Bulk Brain Homogenate Mean (ng/g) MALDI IMS Brain Section Mean (ng/g) Kp,uu Brain Niraparib Control 3847 (769) 658 (111) 532 (89) 0.18 Niraparib BM 2535 (507) 543 (91) 473 (80) 0.14 Niraparib Vehicle BLQ BLQ Olaparib Control 226 (68) 6 (2) BLQ 0.06 Olaparib BM 119 (36) 5 (2) BLQ 0.03 Olaparib Vehicle BLQ BLQ Citation Format: Mark Reid Groseclose, Jeremy Barry, Tinamarie Skedzielewski, Gerald McDermott, Chakravarthi Balabhadrapatruni, Bill Benson, Yongle Pang, David K. Lim, Hoang Tran, Mike Ringenberg, Keyur Gada, Amine Aziez, Elaine Paul, Hasan Alsaid. Differentiation of niraparib and olaparib brain penetration in a mouse brain metastatic tumor model. [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 3582.

Abstract 5045: NTSR1-targeted alpha therapeutic [Ac-225]-FPI-2059 induces growth inhibition in a preclinical colorectal tumor model

Abstract Background: Neurotensin receptor 1 (NTSR1) is overexpressed in multiple cancer indications that include pancreatic, colorectal and prostate cancers, all of which have limited therapeutic treatment options and unmet medical need. Fusion is developing novel targeted alpha therapeutics (TATs) that enable the specific delivery of high energy alpha particles (actinium-225; [225Ac]) to tumor cells while sparing surrounding normal tissues. The alpha radiation released by TATs causes cell damage through the induction of multiple double-stranded DNA breaks leading to tumor cell death. Here, we describe the therapeutic efficacy of an [225Ac]-conjugated, NTSR1 targeting small molecule in a colorectal cancer tumor model. Materials and Methods: CT26 colorectal cancer cells overexpressing murine NTSR1 (mNTSR1) were generated by lentiviral transduction. Selected cells were evaluated for stable mNTSR1 expression by an in vitro radioligand binding assay and subsequently implanted subcutaneously into Balb/c mice for in vivo evaluations. FPI-2056 (parent compound) was radiolabeled with either lutetium-177 ([177Lu]-FPI-2057) or actinium-225 ([225Ac]-FPI-2059). Biodistribution assessment studies were conducted in mice bearing CT26-mNTSR1 tumors dosed intravenously with [177Lu]-FPI-2057. Therapeutic efficacy studies were conducted by intravenous administration of single doses of 0.185 - 5.55 MBq/kg of [225Ac]-FPI-2059 (0.1-3 µCi) to animals bearing CT26-mNTSR1 tumors, followed by tumor growth monitoring for 50 days. Study endpoints included tumor volume measurements and impact on animal health status. Results: Evaluation of [177Lu]-FPI-2057 biodistribution and excretion revealed rapid renal clearance via urine with a clearance from the blood by 24 h. Uptake of [177Lu]-FPI-2057 was detected in the CT26-mNTSR1 tumors with a maximum concentration of 7.0 %ID/g at 6 h post-injection, dropping to 4.6 and 2.8 %ID/g at 24 and 48 h post-injection, with 20-fold higher uptake in the tumor vs. blood levels at both 24 and 48h time points. Therapeutic administration of a single dose of [225Ac]-FPI-2059 resulted in dose-dependent tumor growth inhibition at doses above 1.85 MBq/kg of [225Ac]-FPI-2059 (1 μCi), which translated into increased survival compared to control animals. Conclusion: These results demonstrate that targeted delivery of [225Ac]-FPI-2059 to NTSR1 expressing tumors results in significant growth inhibition and enhanced survival, thereby providing promising preclinical evidence to support the clinical development of [225Ac]-FPI-2059. Citation Format: Saleemulla Mahammad, Jaline Broqueza, Brigitte L. Theriault, John Forbes, Christopher P. Leamon, John Valliant. NTSR1-targeted alpha therapeutic [Ac-225]-FPI-2059 induces growth inhibition in a preclinical colorectal tumor model. [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 5045.

Vitexin suppresses the proliferation, angiogenesis and stemness of endometrial cancer through the PI3K/AKT pathway

Context Endometrial cancer is a common gynecologic malignancy. Vitexin is an active flavonoid compound with an antitumor function. Objective This study elucidated the role of vitexin in endometrial cancer development and clarified the potential mechanism. Materials and methods The toxicity of vitexin (0–80 μM) treatment for 24 h on HEC-1B and Ishikawa cells was tested utilizing the CCK-8 assay. Endometrial cancer cells were divided into vitexin 0, 5, 10, and 20 μM groups. Cell proliferation, angiogenesis and stemness in vitro after treatment with vitexin (0, 5, 10, 20 μM) for 24 h were evaluated using the EdU staining assay, tube formation assay and sphere formation assay, respectively. Twelve BALB/c mice were grouped into control and vitexin (80 mg/kg) groups to monitor tumour growth for 30 days. Results Vitexin suppressed cell viability of HEC-1B (IC50 = 9.89 μM) and Ishikawa (IC50 = 12.35 μM) cells. The proliferation (55.3% and 80% for HEC-1B; 44.7% and 75% for Ishikawa), angiogenesis (54.3% and 78.4% for HEC-1B; 47.1% and 68.2% for Ishikawa) and stemness capacity (57.2% and 87.3% for HEC-1B; 53.4% and 78.4% for Ishikawa) of endometrial cancer cells were inhibited by 10 and 20 μM vitexin. Furthermore, the inhibitory effects of vitexin on endometrial cancer were reversed by PI3K/AKT agonist 740Y-P (20 μM). Moreover, the xenograft tumour experiment lasting for 30 days proved that vitexin (80 mg/kg) blocked tumour growth of endometrial cancer in vivo. Discussion and conclusions Vitexin has therapeutic potential on endometrial cancer, which supports further clinical trials.

The Effects of Omega-3 Polyunsaturated Fatty Acids on Breast Cancer as a Preventive Measure or as an Adjunct to Conventional Treatments

In order to understand how omega-3 polyunsaturated fatty acid (ω-3 PUFA) supplements affect breast cancer prevention and treatment, a systematic review of articles published in the last 5 years in two databases was performed. Of the 679 articles identified, only 27 were included and examined based on five topics, taking into account: the induction type of the breast cancer used in animal models; the characteristics of the induction model by cell transplantation; the experimental design of the ω-3 supplementation-combined or not with a treatment antitumor drug; the fatty acids (FAs) composition used; the analysis of the studies' outcomes. There are diverse and well-established animal models of breast cancer in the literature, with very relevant histological and molecular similarities depending on the specific objective of the study, such as whether the method of tumor induction was transgenic, by cell transplantation, or by oncogenic drugs. The analyses of outcomes were mainly focused on monitoring tumor growth, body/tumor weight, and molecular, genetic, or histological analyses, and few studies evaluated latency, survival, or metastases. The best results occurred when supplementation with ω-3 PUFA was associated with antitumor drugs, especially in the analysis of metastases and volume/weight of tumors or when the supplementation was started early and maintained for a long time. However, the beneficial effect of ω-3 PUFA supplementation when not associated with an antitumor agent remains unclear.