Metastatic Tumor Sites Research Articles

TLR Agonist Therapy of Metastatic Breast Cancer in Mice.

Toll-like receptor (TLR) 7/8 and 9 agonists stimulate an innate immune response that supports the development of tumor-specific immunity. Previous studies showed that either agonist individually could cure mice of small tumors and that when used in combination, they could prevent the progression of larger tumors (>300mm 3 ). To examine whether these agents combined could control metastatic disease, syngeneic mice were challenged with the highly aggressive 66cl4 triple-negative breast tumor cell line. Treatment was not initiated until pulmonary metastases were established, as verified by bioluminescent imaging of luciferase-tagged tumor cells. Results show that combined therapy with TLR7/8 and TLR9 agonists delivered to both primary and metastatic tumor sites significantly reduced tumor burden and extended survival. The inclusion of cyclophosphamide and anti-PD-L1 resulted in optimal tumor control, characterized by a 5-fold increase in the average duration of survival.

Effects of the interactions between platelets with other cells in tumor growth and progression.

It has been confirmed that platelets play a key role in tumorigenesis. Tumor-activated platelets can recruit blood cells and immune cells to migrate, establish an inflammatory tumor microenvironment at the sites of primary and metastatic tumors. On the other hand, they can also promote the differentiation of mesenchymal cells, which can accelerate the proliferation, genesis and migration of blood vessels. The role of platelets in tumors has been well studied. However, a growing number of studies suggest that interactions between platelets and immune cells (e.g., dendritic cells, natural killer cells, monocytes, and red blood cells) also play an important role in tumorigenesis and tumor development. In this review, we summarize the major cells that are closely associated with platelets and discuss the essential role of the interaction between platelets with these cells in tumorigenesis and tumor development.

Abstract 4033: Pan-exon mutant KIT inhibitor DCC-3009 demonstrates tumor regressions in preclinical gastrointestinal stromal tumor models

Abstract Introduction: Gastrointestinal stromal tumors (GISTs) are typically driven by primary mutations in KIT exons 9 or 11. Heterogeneous drug-resistant secondary mutations arise in patients treated with FDA approved KIT inhibitors, including imatinib and sunitinib. Drug resistant secondary mutations are found at multiple regions in the ATP pocket (encoded by exons 13 and 14) or activation switch (encoded by exons 17 and 18) of KIT kinase. In addition, multiple drug-resistant clones can arise within a tumor or in metastatic tumor sites. An inhibitor that can broadly and potently inhibit the spectrum of KIT mutations is highly sought. Ripretinib has been FDA approved as a 4th line treatment for GIST and has broad activity against KIT mutations, including clinical potency in patients with mutations in KIT exons 11, 17, or 18. DCC-3009 was designed as a next generation KIT inhibitor that broadly and potently inhibits primary KIT mutations in exons 9 and 11 and secondary drug-resistant mutations across exons 13, 14, 17, and 18. DCC-3009 is a potent and selective inhibitor in enzyme and cell-based assays, and has demonstrated efficacy in xenograft models driven by drug resistant KIT mutations. Methods: DCC-3009 was tested for inhibition of KIT mutants using standard enzyme and cell-based assays. Levels of phosphorylated KIT were determined by Western blot or ELISA. Proliferation was measured using the fluorescent dye resazurin. KIT mutant xenograft or patient-derived xenograft models were performed at Crown Biosciences or Labcorp, AAALAC accredited facilities, with the approval of Animal Care and Use Committees. Results: In BaF3 cells transfected with KIT mutants, DCC-3009 was shown to potently inhibit the spectrum of known primary and secondary drug-resistant mutations in GIST. The pan-mutant KIT profile of DCC-3009 was shown in vitro to be superior to 2nd and 3rd line standard of care therapies sunitinib and regorafenib. DCC-3009 was selective for KIT when screened against a large panel of kinases. DCC-3009 has optimized pharmaceutical properties for oral administration. In pharmacokinetic/pharmacodynamic studies DCC-3009 achieved sufficient free drug levels to significantly inhibit drug-resistant KIT mutants for 12 hr post dose. In xenograft studies, treatment with DCC-3009 twice daily led to tumor regression in drug-resistant models with KIT exon 9/13, 11/13 or 11/17 mutations. Conclusions: DCC-3009 is a pan-exon mutant KIT inhibitor exhibiting high potency in KIT mutants in pre-clinical models spanning exons 9, 11, 13, 14, 17 and 18. In vivo, DCC-3009 exhibited efficacy in drug-resistant models with KIT exon 9/13, 11/13 or 11/17 mutations. Based on this profile, DCC-3009 has entered formal preclinical development. Citation Format: Bryan D. Smith, Subha Vogeti, Timothy M. Caldwell, Hanumaiah Telikepalli, Yu Mi Ahn, Gada Al-Ani, Stacie L. Bulfer, Andrew Greenwood, Cale L. Heiniger, Joshua W. Large, Cynthia B. Leary, Wei-Ping Lu, Kylie Luther, William C. Patt, Max D. Petty, Yeni K. Romero, Forrest A. Stanley, Kristen L. Stoltz, Daniel C. Tanner, Sihyung Yang, Yu Zhan, Bertrand Le Bourdonnec, Daniel L. Flynn. Pan-exon mutant KIT inhibitor DCC-3009 demonstrates tumor regressions in preclinical gastrointestinal stromal tumor models. [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 4033.

Abstract 1289: The role of mutant p53-mediated mechanisms in modulating the tumor microenvironment and promoting lung metastasis

Abstract INTRODUCTION/METHODS: The multistep process of the tumor invasion-metastasis cascade is a major barrier to effective therapy. Mutations of the tumor suppressor p53 are frequently detected in esophageal squamous cell carcinoma (ESCC) cases, which in turn correlate with high metastatic rates. To understand the mutant p53-mediated mechanisms in promoting ESCC metastasis, we conducted RNA-Seq, ChIP-Seq and cytokine array on isogenic primary and metastatic tumor cells harvested from our mouse model of esophageal cancer, L2-Cre; LSL-Trp53R172H; Rosa26LSL-YFP, in which we have identified macrophage colony stimulating factor 1 (CSF1) to be a direct gene target of p53-R172H. We have targeted CSF1/CSF1R signaling to assess its pro-tumorigenic roles. In addition to studying tumor intrinsic mechanisms, we have established multiplex immunofluorescence and flow cytometry approaches to characterize the changes in tumor microenvironment (TME). Overall, this study aims to investigate the role and mediators of CSF1 signaling through its cognate receptor CSF1R by which missense p53 mutations can promote lung metastasis. RESULTS/DISCUSSION: We demonstrate that metastatic ESCC has increased Csf1 expression compared to primary tumors and this is dependent upon p53 mutation status, which is reinforced by TCGA data and patient-derived tissue microarrays (TMAs). The TMA analysis also suggested that CSF1 expression is higher in tumors that are poorly differentiated. Furthermore, through the proximity ligation assay (PLA), we have identified that bromodomain and extra-terminal (BET) protein BRD4, which binds to acetylated histones to regulate transcription interacts with p53-R172H at higher levels compared to wild-type p53 to induce Csf1 expression. We show that the BRD4/CSF1/CSF1R signaling axis fosters tumor invasion, subcutaneous tumor growth and metastatic burden in lungs in a mutant p53 background. In accordance, targeting CSF1/CSF1R signaling reduces phosphorylation of STAT3 in the metastatic tumors, which suggests a mechanism for increased invasiveness and metastasis. In parallel, upon inhibiting this axis, we have identified decreased infiltration of F4/80+CD163+ and F4/80+CD206+ M2-polarized macrophages, as well as CD31+ endothelial cells, and increased number of CD3+CD8+ cytotoxic T-cells at the metastatic tumor sites, indicating that the CSF1/CSF1R pathway plays a critical role in shaping the pro-metastatic and immunosuppressive TME. Finally, analysis of squamous cell carcinoma (SCC) datasets reveals that specific p53 mutations are associated with differential survival rates and CSF1 expression. CONCLUSION: We have demonstrated novel roles and mechanisms of mutant p53-dependent CSF1-CSF1R signaling pathway in fostering ESCC tumor invasion and lung metastasis that may be applicable to other SCCs. We believe this can open up new avenues for therapeutic applications. Citation Format: Gizem Efe, Katherine M. Cunningham, Qiaosi Tang, Kensuke Sugiura, Karen Dunbar, Kensuke Suzuki, Lois Resnick-Silverman, Alison M. Taylor, James J. Manfredi, Carol L. Prives, Anil K. Rustgi. The role of mutant p53-mediated mechanisms in modulating the tumor microenvironment and promoting lung metastasis [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 1289.

Abstract 820: Photo-activatable ON-OFF microparticles for on-demand pulsatile drug delivery in a breast cancer model

Abstract The ability to treat tumors with high efficacy in a local microenvironment, while mitigating the side effects of systemic toxicity is an area of active investigation. Towards this goal, designing a minimally-invasive local therapeutic modality, with a controlled, pulsatile drug release mechanism is an attractive proposition. In this study, a near-infrared (NIR) photo-activatable microparticle was developed for localized, pulsatile delivery of encapsulated anticancer drugs into the tumor with simultaneous thermal ablation, with controlled ON-OFF thermal cycles using NIR laser irradiation. The microparticles were fabricated using a poly(caprolactone) (PCL) matrix, containing 2D molybdenum disulfide (MoS2) nanosheets as the NIR-responsive photothermal agent, and doxorubicin or violacein, as hydrophilic and hydrophobic model anticancer drugs, respectively. Cubic microparticles of 200 µm size were fabricated by casting a polymer-MoS2-drug film on PDMS mold with loading efficiency of 2 μg MoS2 per particle, and 0.2-8 μg of drug per particle. A cytotoxicity assay was performed to test the efficacy of the loaded microparticles in reducing the viability of 4T1 murine-derived cell culture, using the ON-OFF laser switch mechanism. In order to select a suitable laser power and duration of treatment for in vivo studies, a Machine Learning algorithm based on a Random Forest classifier was trained, to arrive at the optimal treatment conditions: 0.4-0.5 W/cm2 of laser power at 808 nm, for 3 cumulative minutes of laser irradiation, to reach the target temperature of 50 °C, which activates PCL melting and subsequent drug release. The effect of pulsatile treatment was studied in vivo in a murine-derived 4T1 subcutaneous mouse model of breast cancer, using this photo-activatable ON-OFF switch mechanism, for up to 3 cycles of laser irradiation. As control groups, tumor only (no treatment), laser only (no drug or microparticle), drug only (no laser or microparticle), microparticles only (no drug or laser), and microparticles with laser (no drug) were studied. The cohorts receiving the photo-activated 3-cyclic treatment for both drugs showed increased median survival up to 40 days post tumor induction, compared to a median survival of 16 days for the control groups. Although one laser cycle was enough to trigger drug release, there was a significant shrinkage in the tumor volume noticed with 3 cycles, with eventual scarring and shedding of the tissue with no evidence of residual tumor at the primary site. While this cyclic drug release modality is very effective at treating the primary site, subsequent histopathology revealed that the aggressive tumor in some animals had metastasized to the liver and other organs. More work is ongoing to design better targeted approaches to deliver the drug-loaded microparticles to the site of metastatic tumors, to increase the survival prospects of this disease. Citation Format: Maria Kanelli, Neelkanth M. Bardhan, Morteza Sarmadi, Shahad Alsaiari, William Rothwell, Apurva Pardeshi, Angela M. Belcher, Ana Jaklenec, Robert S. Langer. Photo-activatable ON-OFF microparticles for on-demand pulsatile drug delivery in a breast cancer 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 820.

Abstract 6117: Dose, dose rate, and linear energy transfer influence tumor immunologic and DNA damage response following alpha- and beta-emitting radionuclides

Abstract Background: The low response rate to immunotherapies in poorly immunogenic cancers highlights the potential for combination therapies that propagate an anti-tumor response in metastatic settings. Targeted radionuclide therapy (TRT) can deliver radiation to metastatic tumor sites. In preclinical studies, combining low dose TRT with immune checkpoint blockade augments the anti-tumor immune response, promoting the immune susceptibility of metastatic disease sites. Radionuclides differ in their physical properties such as emission type, linear energy transfer (LET), half-life, and tissue range. In this study, clinically relevant α- and β-emitting radionuclides (225Ac, 90Y, 177Lu) were compared in vitro in MOC2 head and neck squamous cell carcinoma and B78 melanoma. We hypothesized that the unique physical properties of radionuclides would differentially impact the quantity and accumulation over time of double stranded DNA breaks, mirroring the timing of effects on tumor cell immune susceptibility markers. Methods: MOC2 or B78 cells were grown in culture and treated with external beam radiation (EBRT), or culture media containing 90Y, 177Lu, or 225Ac delivering continuous radiation at activity levels using GEANT4 Monte Carlo to deliver 12 Gy (MOC2) and 4 Gy (B78) to the cell monolayer. Cells were harvested, and cDNA was isolated for RT-qPCR one, three, and seven days after the start of irradiation. Additional cells treated in this manner were fixed and stained with DAPI/γ-H2AX antibody for confocal microscopy at the same timepoints. γ-H2AX foci/cell were quantified manually using ImageJ. Results: γ-H2AX foci counts/cell increased significantly and accumulated over time following treatment with 225Ac, but not with 90Y or 177Lu. EBRT, 90Y, and 225Ac, but not 177Lu, upregulated expression of immune response associated genes (Fas, Pdl1, Mhc1) compared to non-irradiated controls. In cells treated with EBRT every 24h at doses mimicking the interval dose delivered by continuous exponential decay of 90Y and 225Ac, but not 177Lu, the time course and magnitude of Fas and Pdl1 expression phenocopied that of these radionuclides. Conclusions: DNA damage accumulation and the timing of immunomodulation are modified by the dose, dose-rate, and LET of radiation emitted from a given radionuclide. Understanding the effects of radionuclide therapies on cancer cell immunogenicity could enable rational design of clinical trials that investigate the integration of TRT and immunotherapies into the clinical care of patients with metastatic cancers. Citation Format: Julia Sheehan-Klenk, Caroline P. Kerr, Thanh P. Nguyen, Joseph J. Grudzinski, David Adam, Maria Powers, Raghava N. Sriramaneni, Paul A. Clark, Reinier Hernandez, Bryan Bednarz, Jamey P. Weichert, Zachary S. Morris. Dose, dose rate, and linear energy transfer influence tumor immunologic and DNA damage response following alpha- and beta-emitting radionuclides [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 6117.

Stereotactic ablative body radiotherapy with a central high dose using CyberKnife for metastatic lung tumors

BackgroundThe CyberKnife system features a robotically-positioned linear accelerator to deliver real-time image-guided stereotactic ablative body radiotherapy (SABR). It achieves steep dose gradients using irradiation from hundreds of different directions and increases the central dose of the gross tumor volume (GTV) without increasing the marginal dose to the planning target volume. We evaluated the effectiveness and safety of SABR with a central high dose using CyberKnife for metastatic lung tumors.MethodsA total of 73 patients with 112 metastatic lung tumors treated with CyberKnife were retrospectively analyzed. Local control, progression-free survival, and overall survival were calculated using the Kaplan–Meier method. The median age was 69.2 years. The most common primary sites were the uterus (n = 34), colorectum (n = 24), head and neck (n = 17), and esophagus (n = 16). For peripheral lung tumors, the median radiation dose was 52 Gy in 4 fractions, whereas for centrally located lung tumors, it was 60 Gy in 8–10 fractions. The dose prescription was defined as 99% of the solid tumor components of the GTV. The median maximum dose within the GTV was 61.0 Gy. The GTV and planning target volume were enclosed conformally by the 80% and 70% isodose lines of the maximum dose, respectively. The median follow-up period was extended to 24.7 months; it was 33.0 months for survivors.ResultsThe 2-year local control, progression-free survival, and overall survival rates were 89.1%, 37.1%, and 71.3%, respectively. Toxicities of grade ≥ 2 were noted as grade 2 and 3 radiation pneumonitis in one patient each. The two patients with grade 2 or higher radiation pneumonitis had both received simultaneous irradiation at two or three metastatic lung tumor sites. No toxicity of grade ≥ 2 was observed in patients with metastasis in one lung only.ConclusionsSABR with a central high dose using CyberKnife for metastatic lung tumors is effective with acceptable toxicity.Trial registrationNumber: 20557, Name: Stereotactic ablative radiotherapy using CyberKnife for metastatic lung tumor, URL: http://www.radonc.med.osaka-u.ac.jp/pdf/SBRT.pdf, Date of registration: April 1, 2021 (retrospectively registered), Date of enrollment: May 1, 2014.

Bone marrow niche chemoprotection of metastatic solid tumors mediated by CYP3A4.

The bone/bone marrow is one of the most common sites for metastatic solid tumors. Moreover, the tumor microenvironment is an essential part of cancer homeostasis. Previously, it was shown that cytochrome P450 enzymes (CYPs) are present in the bone marrow (BM) microenvironment, particularly in the mesenchymal stroma cells, at levels comparable to those of hepatocytes. It was found that the CYPs play important roles in nurturing and maintaining normal hematopoietic stem cells as well as multiple myeloma and leukemia cells, including protecting them from toxic insults. It was hypothesized that the CYPs in the BM microenvironment might play a similar role in solid tumors metastatic to bone. The interaction between the BM microenvironment and malignant cells that routinely metastasize to the bone (lung, breast, and prostate cancer) was modeled. Via genetic engineering and pharmacological approaches, the role of stromal cytochrome P450 3A4 (CYP3A4) in drug resistance promoted by the BM microenvironment in niche-cancer models in vitro and in vivo was interrogated. BM stroma protected prostate, breast, and lung cancer cells from cytotoxic chemotherapy. Stromal CYP3A4 was at least partially responsible for this protection in vitro and in vivo. Moreover, inhibiting CYP3A4 with clarithromycin overcame the stroma-mediated chemoresistance toward prostate, breast, and lung cancer cells. These results suggest that, similar to observations from hematologic malignancies, the BM microenvironment, through expression of CYPs, creates a sanctuary site from chemotherapy for metastatic solid tumors. Targeting these sanctuaries holds promise for eradicating bone metastasis in solid tumors.

SREKA-targeted liposomes for highly metastatic breast cancer therapy

Chemotherapy is still a leading therapeutic approach in various tumor types that is often accompanied by a poor prognosis because of metastases. PEGylated liposomes with CREKA targeting moiety are well-known therapeutic agents, especially in highly metastatic experimental models. CREKA specifically targets tumor-associated ECM, which is present at the primary, as well as metastatic tumor sites. To better understand the function of the targeting moieties, we decided to design various liposome formulations with different amounts of targeting moiety attached to their DSPE-PEG molecules. Moreover, a new tumor-homing pentapeptide (SREKA) was designed, and a novel conjugation strategy between SREKA and DSPE-PEGs. First, the in vitro proliferation inhibition of drug-loaded liposomes and the cellular uptake of their cargo were investigated. Afterward, liposome stability in murine blood and drug accumulation in different tissues were measured. Furthermore, in vivo tumor growth, and metastasis inhibition potencies of the different liposome formulations were examined. According to our comparative studies, SREKA-liposomes have a uniform phenotype after formulation and have similar characteristics and tumor-homing capabilities to CREKA-liposomes. However, the exchange of the N-terminal cysteine to serine during conjugation results in a higher production yield and better stability upon conjugation to DSPE-PEGs. We also showed that SREKA-liposomes have significant inhibition on primary tumor growth and metastasis incidence; furthermore, increase the survival rate of tumor-bearing mice. Besides, we provide evidence that the amount of targeting moiety attached to DSPE-PEGs is largely responsible for the stability of liposomes, therefore it plays an important role in toxicity and targeting.

Abstract B014: Mutant p53-mediated CSF1/CSF1R signaling promotes tumor invasion and lung metastasis in esophageal squamous cell carcinoma

Abstract Introduction: Mutations of the tumor suppressor p53 are detected up to 80% of esophageal squamous cell carcinoma (ESCC) cases, which in turn correlate with high metastatic rates and poor prognosis. To understand the mutant p53-mediated mechanisms in promoting ESCC metastasis, we conducted RNA-Seq and cytokine array on isogenic primary and metastatic tumor cells harvested from our mouse model of esophageal cancer harboring Trp53R172H/-, in which we have identified Colony stimulating factor 1 (Csf1) to be upregulated. Our goal is to investigate the role and mediators of CSF1 signaling through its cognate receptor CSF1R by which missense p53 mutations can promote tumor invasion and lung metastasis in ESCC. Methods: We have utilized novel L2-Cre; LSL-Trp53R172H; Rosa26LSL-YFP mice and isolated tumor cells to model metastatic ESCC. We conducted ChIP-Seq analysis for p53 on tumor cells derived from lung metastases in our mouse models. Furthermore, we have genetically and pharmacologically targeted CSF1/CSF1R signaling axis to assess its role in ESCC tumor invasion and lung metastasis. In addition to studying tumor intrinsic mechanisms, we have established quantitative multiplex immunofluorescence (qmIF) and flow cytometry approaches to characterize the changes in tumor microenvironment (TME). Results And Discussion: We demonstrate that metastatic ESCC has increased Csf1 expression compared to primary tumors, and this is dependent upon p53 mutation status, which is reinforced by the TCGA data and patient-derived tissue microarrays (TMAs). The overlay of the RNA-Seq with the ChIP-Seq analysis indicates that Csf1 is a direct gene target of p53-R172H with enriched binding motifs. Furthermore, based on the proximity ligation assay (PLA) with murine ESCC tumor cells, bromodomain and extra-terminal motif (BET) protein BRD4 interacts with p53-R172H to induce Csf1 expression. We show that the BRD4-CSF1 axis fosters tumor invasion, subcutaneous tumor growth and metastatic burden in a mutant p53 background. In accordance, upon inhibiting this signaling pathway in the tail-vein injection lung metastasis models, we have identified reduced expression of CD31+ cells and decreased infiltration of F4/80+CD163+ and F4/80+CD206+ M2-polarized macrophages at the metastatic tumor sites, indicating that the CSF1/CSF1R pathway plays a critical role in shaping the pro-metastatic and immunosuppressive TME. Finally, analysis of the esophageal cancer datasets reveals that specific p53 mutations are associated with markedly differential overall survival rates and Csf1 expression. Based upon these results, we are generating human and murine esophageal cells with distinct DNA contact and conformational p53 mutations via base editing. Conclusion: We have demonstrated novel roles and mechanisms of mutant p53-dependent CSF1/CSF1R signaling pathway in fostering ESCC tumor invasion and lung metastasis that may be applicable to other squamous cell cancers. We believe this can open up new avenues for therapeutic applications. Citation Format: Gizem Efe, Qiaosi Tang, Katherine M. Cunningham, Kensuke Sugiura, Karen Dunbar, Kausik Regunath, Kensuke Suzuki, Andres J. Klein-Szanto, Lois Resnick-Silverman, James J. Manfredi, Carol L. Prives, Anil K. Rustgi. Mutant p53-mediated CSF1/CSF1R signaling promotes tumor invasion and lung metastasis in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B014.

Radiation to all macroscopic sites of tumor permits greater systemic antitumor response to in situ vaccination

BackgroundThe antitumor effects of external beam radiation therapy (EBRT) are mediated, in part, by an immune response. We have reported that a single fraction of 12 Gy EBRT combined with...

Outcomes of Sling Procedure Using a Free Vascularized Fibular Graft After Resection of the Proximal Humerus.

The proximal humerus is a common site for both primary and metastatic bone tumors. Although various methods have been developed for reconstruction following resection of the proximal humerus, a consensus on which technique is best has not been established. We focused on the sling procedure using a free vascularized fibular graft (FVFG) and conducted what we believe to be the largest retrospective study of patients to undergo this surgery to date. We retrospectively reviewed the data of 19 patients who underwent the sling procedure with use of an FVFG at our hospital between 1998 and 2022. The median age was 20 years, and the median follow-up duration was 63.1 months. Surgical data, oncological outcomes, the postoperative course, complications, and functional outcomes as measured with use of the Musculoskeletal Tumor Society (MSTS) score were thoroughly reviewed. The median operative duration was 555 minutes, and the median blood loss was 374 mL. The median length of the bone defect was 17.0 cm, and the median length of the graft was 20.0 cm. With respect to oncological outcomes, 9 patients were continuously disease-free, 9 patients had no evidence of disease, and 1 patient was alive with disease. Bone union was present in 13 of the 17 patients for whom it was evaluable. The median time to bone union was 4 months. Graft growth was observed in 2 pediatric patients. Postoperative fracture was a major complication at the recipient site. The incidence of pseudarthrosis significantly increased when the FVFG could not be inserted into the remaining humeral bone or was split in half (p = 0.002). Although a few patients demonstrated peroneal nerve palsy at the donor site, the symptom was temporary. The overall functional outcome was favorable, with an average MSTS score of 66.9%. The sling procedure demonstrated a low complication rate and a favorable functional outcome overall. Therefore, we believe that this procedure is a useful reconstruction method for patients in a broad age range who have a wide defect of the proximal humerus. Therapeutic Level IV. See Instructions for Authors for a complete description of levels of evidence.

A 3D, Compartmental Tumor-Stromal Microenvironment Model of Patient-Derived Bone Metastasis

Bone is a frequent site of tumor metastasis. The bone-tumor microenvironment is heterogeneous and complex in nature. Such complexity is compounded by relations between metastatic and bone cells influencing their sensitivity/resistance to chemotherapeutics. Standard chemotherapeutics may not show efficacy for every patient, and new therapeutics are slow to emerge, owing to the limitations of existing 2D/3D models. We previously developed a 3D interface model for personalized therapeutic screening, consisting of an electrospun poly lactic acid mesh activated with plasma species and seeded with stromal cells. Tumor cells embedded in an alginate-gelatin hydrogel are overlaid to create a physiologic 3D interface. Here, we applied our 3D model as a migration assay tool to verify the migratory behavior of different patient-derived bone metastasized cells. We assessed the impact of two different chemotherapeutics, Doxorubicin and Cisplatin, on migration of patient cells and their immortalized cell line counterparts. We observed different migratory behaviors and cellular metabolic activities blocked with both Doxorubicin and Cisplatin treatment; however, higher efficiency or lower IC50 was observed with Doxorubicin. Gene expression analysis of MDA-MB231 that migrated through our 3D hybrid model verified epithelial-mesenchymal transition through increased expression of mesenchymal markers involved in the metastasis process. Our findings indicate that we can model tumor migration in vivo, in line with different cell characteristics and it may be a suitable drug screening tool for personalized medicine approaches in metastatic cancer treatment.

Theranostics in prostate cancer

SummaryThis review summarizes current evidence of “theranostics” for patients with prostate cancer. Prostate-specific membrane antigen (PSMA) is a glycoprotein which can be labeled with positron-emitting tomographic (PET) tracers like fluorine-18, gallium-68, or copper-64 and can be labeled with beta emitters like lutetium-177 to treat patients with metastatic castration-resistant prostate cancer (mCRPC). PSMA PET/CT has been shown to be superior to computer tomography (CT) and bone scintigraphy in accuracy, sensitivity, and specificity in the evaluation of metastatic tumor sites and may change treatment management. However, outcome studies showing an improvement in progression-free survival (PFS) and/or overall survival (OS) after management change is missing. PSMA PET/CT is highly recommended in prostate cancer patients with biochemical recurrence (PSA > 0.2 ng/ml). Patients presenting with high PSMA expression in PSMA PET/CT may be favorably treated with 177lutetium-617. In the TheraP study, a multicenter phase 2 trial, patients with mCRPC were randomly assigned to receive either 177lutetium-617 or cabazitaxel. The results indicate fewer treatment-related adverse events in patients treated with 177lutetium-617, an improvement in PSA response, but no improvement in OS after 36 months follow-up. In the VISION trial, a phase 3 international study, patients with mCRPC were randomly assigned to receive either 177lutetium-617 and standard of care (SOC) or SOC alone. At 12 months, radiographic PFS or PSA-PFS was 18% in patients treated with 177lutetium-617 and 3% in patients treated with SOC alone. The real status of 177lutetium-617 in the sequel of treatment regimens remains unclear and additional studies are therefore warranted.

864 ISOLATED CARDIAC METASTASIS FROM CARCINOMA OF THE CERVIX: RARE CASE OF ACS-NSTE. CASE REPORT

Abstract Introduction The heart is a rare site of tumor metastasis. Although rarely, cardiac metastases may be secondary to a primary intra-abdominal tumor. Aim we report the case of a 60-years-old woman, ante-mortem finding of an isolated cardiac metastasis from cervical carcinoma presented as ACS-NSTE, who was evaluated in our Division of Cardiology. Materials and methods electrocardiogram, transthoracic echocardiography, angioTC, cardiac surgery and histopathologic analysis were performed. Results electrocardiogram showed incomplete right branch block with diffuse negative T waves. On the echocardiogram evidence of a voluminous hypo-isoechoic formation almost completely occupying the cavity of the right ventricule (RV) which appeared dilated and dysfunctional; D-shape aspect of the left ventricule as from the overload of the right sections and with moderate pericardial effusion, mildly reduced ejection fraction (EF50%Q). An urgent Angio-CT was performed and revealed a voluminous solid neoformation with inhomogeneous content and progressive contrast enhancement in the right ventricule. Following admission, the patient's haemodynamic status gradually worsened. Therefore, urgent cardiac surgery was performed. After drainage of abundant serum/haematic effusion, probably neoplastic infiltration of RV was seen. Therefore, given the extension it was decided to perform only multiple biopsies. Histopathological examination revealed moderately differentiated squamous cell carcinoma, as from metastasis from K. cervix. Conclusions there is no standardized therapy for the treatment of cardiac metastases, it is necessary to evaluate on a case-by-case basis. The most important prognostic factors are the obstructive effect of the intracardiac mass and the extent of any pericardial effusion. In fact, they can adversely affect hemodynamic stability.

Nanoadjuvants Actively targeting lymph node conduits and blocking tumor invasion in lymphatic vessels.

Great efforts have been made to manipulate nanoparticles (NPs) with a diameter of 10-100nm to passively target lymph nodes (LNs) to magnitude anti-tumor activity of T cells. However, no attention has been paid to increasing the retention of NPs with active affinity in order to induce a prolonged release of antigens or molecular adjuvants in the LNs mattering the immune response. Here, we formulated two NPs encapsulated with imiquimod (IMQ), a TLR7/8 agonist, and paclitaxel (PTX) and further modified them with tannic acid (TA), respectively, to generate IMQ NP and PTX NP with a final diameter of approximately 40nm. Attributing a strong affinity of TA molecules to the elastin of LN conduits, the TA modified IMQ NPs can bypass the gaps in the layer of lymphatic endothelial cells and enter the paracortex through the lymph node capsule-associated (LNC) conduits. Similarly, the TA modified PTX NPs increased delivery of PTX to the metastatic tumor site in LNs, where the tumor-associated antigens were released and presented by conduits-lining dendritic cells to activate T cells. Thus, the NPs with deposition to LN conduits showed excellent performance in preventing lymphovascular invasion of triple-negative breast cancer cells and lung metastasis thereafter. On the contrary, the NPs without TA flowed through the subcutaneous sinus existing LNs directly by efferent lymphatic vessels showing relatively poor therapeutic outcomes. This study reveals that TA may mediate the long retention of antigens and molecular adjuvants to be delivered to deep LNs for developing potent vaccination technology.

Multi-scale characterization of tumor-draining lymph nodes in resectable lung cancer treated with neoadjuvant immune checkpoint inhibitors

SummaryBackgroundRegional lymph node (LN) acts as a pivotal organ for antitumor immunity. Paradoxically, tumor-draining LNs (TDLNs) are usually the first site of tumor metastasis in lung cancer. It is largely unknown about the association between the status of TDLNs and the response of primary tumor beds to immune checkpoint inhibitors (ICIs) in lung cancer patients. Also, studies characterizing the TDLNs in response to ICIs are scarce.MethodsWe characterized and compared the radiological, metabolic (18F-FDG) and pathologic responses between primary tumor beds and paired TDLNs (invaded/non-invaded) from 68 lung cancer patients who underwent neoadjuvant ICIs plus surgery. Additionally, we performed the spatial profiling of immune and non-immune cells within TDLNs using multiplexed immunofluorescence. Therapy responses (e.g., pathologic complete (pCR) or major response (MPR)) of primary lung tumor beds and paired TDLNs were investigated separately.FindingsWe observed that responses of TDLNs to ICIs markedly differ from their paired primary lung tumors regarding the radiological, metabolic (18F-FDG uptake), and pathologic alterations. Neoadjuvant ICIs therapy specifically decreased 18F-FDG-reflected metabolic activity in the primary tumor beds with pCR/MPR but not their TDLNs counterparts. Furthermore, the presence of invaded TDLNs was associated with poor pathologic responses in the matched primary tumor beds and predictive of rapid post-treatment tumor relapse. Spatial profiling demonstrated exclusion of T cell infiltrates within the metastatic lesions of invaded TDLNs, and diminished multiple immune and non-immune compositions in non-involved regions surrounding the metastatic lesions.InterpretationThese results provide the first clinically-relevant evidence demonstrating unique response patterns of TDLNs under ICIs treatment and revealing the underappreciated association of TDLNs status with the response of their paired primary tumors to ICIs in lung cancer.FundingThis work was supported by the National Natural Science Foundation of China (82072570 to F. Yao; 82002941 to B. Sun), the excellent talent program of Shanghai Chest Hospital (to F.Y), the Basic Foundation Program for Youth of Shanghai Chest Hospital (2021YNJCQ2 to H.Yang), and the Innovative Research Team of High-level Local Universities in Shanghai (SHSMU-ZLCX20212302 to F. Yao).

Nanomaterial-Based Drug Delivery System Targeting Lymph Nodes.

The lymphatic system plays an indispensable role in humoral balance, lipid metabolism, and immune regulation. The lymph nodes (LNs) are known as the primary sites of tumor metastasis and the metastatic LNs largely affected the prognosis of the patiens. A well-designed lymphatic-targeted system favors disease treatment as well as vaccination efficacy. In recent years, development of nanotechnologies and emerging biomaterials have gained increasing attention in developing lymph-node-targeted drug-delivery systems. By mimicking the endogenous macromolecules or lipid conjugates, lymph-node-targeted nanocarries hold potential for disease diagnosis and tumor therapy. This review gives an introduction to the physiological functions of LNs and the roles of LNs in diseases, followed by a review of typical lymph-node-targeted nanomaterial-based drug-delivery systems (e.g., liposomes, micelles, inorganic nanomaterials, hydrogel, and nanocapsules). Future perspectives and conclusions concerned with lymph-node-targeted drug-delivery systems are also provided.

Abstract 1596: Genomic heterogeneity as a barrier to precision oncology in urothelial cancer

Abstract Background: Precision oncology relies on the accurate molecular characterization of individual cancer patients at the time of treatment initiation. However, tumor molecular profiles are not static, and cancers continually evolve because of ongoing mutagenesis and clonal selection, with actionable genomic alterations potentially gained or lost during disease progression. Methods: To define the concordance of potentially actionable genomic alterations between primary and metastatic disease sites in patients with urothelial cancer and to identify drivers of metastatic disease progression, we performed an integrated analysis of clinical and genomic data from 2,199 urothelial carcinoma patients (2,732 tumor and 331 plasma cfDNA samples) prospectively profiled at Memorial Sloan Kettering Cancer Center (MSKCC) from 2014 to 2021. Paired primary and metastatic tumor samples from individual patients were sequenced with MSK-IMPACT and/or whole-exome sequencing (WES). Plasma samples were analyzed using the MSK-ACCESS cfDNA platform. Results: Among potentially actionable mutations (defined as OncoKB level 1-4), ERBB2 and ARID1A were associated with higher tumor grade and stage (p-value <0.001, p-value = 0.03 respectively). WES analysis of primary and metastatic tumor sites was consistent with early branched evolution with on average 42% of mutations shared between disease sites. Among chromatin-modifying genes, ARID1A has a discordant rate of 15.8%. In the 6 discordant cases, ARID1A mutation was seen exclusively in the metastatic tumor samples. This suggests a role for ARID1A mutations in metastatic disease progression. While known and likely oncogenic mutations were more likely to be concordant than variants of unknown functional significance, we observed a high degree of mutational discordance among potentially actionable genomic alterations with 23% discordant between primary and metastatic disease sites. 24% of mutations were exclusively identified by plasma cell-free DNA sequencing. Actionable mutations in cell-free DNA were more likely to be concordant with metastatic tumor biopsies than tumor tissue collected from the primary tumor site. Conclusions: In sum, our analysis of patient-matched primary and metastatic urothelial carcinomas revealed a high degree of lesion-to-lesion genomic heterogeneity that may be a barrier to precision oncology approaches for this disease. Our data also provide a rationale for the use of cell-free DNA sequencing to guide targeted therapy selection in patients with metastatic urothelial cancer. Citation Format: Ziyu Chen, Timothy N. Clinton, Soonbum Park, Andrew T. Lenis, Mark Donoghue, Jonathan E. Rosenberg, Bajorin F. Dean, Min Y. Teo, Bernard H. Bochner, Irina Ostrovnaya, Eugene J. Pietzak, Gopakumar V. Iyer, Sizhi Gao, Wenhuo Hu, Michael M. Shen, Michael F. Berger, Hikmat A. Al-Ahmadie, Corinne T. Abate-Shen, David B. Solit. Genomic heterogeneity as a barrier to precision oncology in urothelial 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 1596.

Abstract 5531: PD-1 blockades plus targeted therapy and cytotoxic drugs as conversion therapy in unresectable metastatic colorectal cancer (mCRC): A real-world experience

Abstract Background: Resection of metastatic tumor site has been proven beneficial for survival in mCRC, thus the need for conversion therapy of mCRC is strong. Anti-PD-1 antibody has presented durable clinical response in mismatch repair deficient (dMMR)/microsatellite instable (MSI-H) mCRC as first-line treatment while its efficacy in microsatellite stable (MSS) mCRC is still controversial. Combination of anti-PD-1 and targeted therapy or chemotherapies was regarded as potential regimens in MSS mCRC. Moreover, it is fully elucidated that immune checkpoint inhibitor used in initial therapy would have higher probability to achieve treatment response. We retrospectively analyzed the efficacy of anti-PD-1 plus targeted drugs and cytotoxic drugs as conversion/first-line therapy in fifteen untreated mCRC. Methods: Thirteen MSS and two MSI-H mCRC were included. All patients received at least one cycle of anti-PD-1 (Pembrolizumab, Sintilimab or Toripalimab, q3w) combined with bevacizumab or cetuximab plus chemotherapy (FOLFOX or FOLFOXIRI regimens). Results: 8 patients were RAS-mutant and 1 patient harbored BRAF-V600E. 13 patients had liver metastasis, 3 had lung metastasis and 3 had retroperitoneal lymph metastasis. 4 patients, including 2 MSI-H patients and 2 MSS patients had complete response (CR), 8 patients had partial response (PR), 2 patients had stable disease (SD) and 1 patient had progression disease (PD). The conversion rate was 80% (12/15), including 10 patients received resection or thermal ablation of metastatic tumor and 2 patients achieved CR. 9 patients were R0 resection and 10 patients achieved NED (no evidence of disease) status. G3-4 treatment-related adverse events (AEs) occurred in 1 patient (neutropenia, n=1). No patient discontinued treatment due to adverse events. Conclusions: The triplet regimen of anti-PD-1, bevacizumab or cetuximab and chemotherapy was safe and tolerant and showed encouraging anti-tumor activities in MSS mCRC. Our experience suggested the clinical value of PD-1 blockades as initial treatment in MSS mCRC. Citation Format: Ling Huang, Liying Zhao, Xinyi Liu, Mengli Huang. PD-1 blockades plus targeted therapy and cytotoxic drugs as conversion therapy in unresectable metastatic colorectal cancer (mCRC): A real-world experience [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 5531.