Reduction In Metastatic Burden Research Articles

Simulating the Effect of Removing Circulating Tumor Cells (CTCs) from Blood Reveals That Only Implantable Devices Can Significantly Reduce Metastatic Burden of Patients.

Circulating tumor cells (CTCs) are cells that have separated from a solid cancerous lesion and entered the bloodstream. They play a crucial role in driving the metastatic spread to distant organs, which is the leading cause of cancer-related deaths. Various concepts for blood purification devices aiming to remove CTCs from the blood and prevent metastases have been developed. Until now, it is not clear if such devices can indeed reduce new metastasis formation in a significant way. Here, we present a simple theoretical model of CTCs in the bloodstream that can be used to predict a reduction in metastatic burden using an extracorporeal or intracorporeal blood purification device. The model consists of a system of ordinary differential equations that was numerically solved and simulated. Various simulations with different parameter settings of extracorporeal and intracorporeal devices revealed that only devices implanted directly in tumor-draining vessels can reduce the metastatic burden significantly. Even if an extracorporeal device is used permanently, the reduction in metastases is only 82%, while a permanently operating implanted device in the tumor-draining vessel would achieve a reduction of 99.8%. These results are mainly due to the fact that only a small fraction of CTCs reaches peripheral circulation, resulting in a proportionally small amount of purified blood in extracorporeal devices.

Nonlinear progression during the occult transition establishes cancer lethality.

Cancer screening is based upon a linear model of neoplastic growth and malignant progression. Yet, historical observations suggest that malignant progression is uncoupled from growth which may explain the paradoxical increase in early-stage breast cancer detection without a dramatic reduction in metastatic burden. Here we lineage trace millions of genetically transformed field cells and thousands of screen detectable and symptomatic tumors using a cancer rainbow mouse model of HER2+ breast cancer. Transition rates from field cell to screen detectable tumor and then to symptomatic tumors were estimated from a dynamical model of tumor development. Field cells are orders of magnitude less likely to transition to a screen detectable tumor than the subsequent transition of a screen detectable tumor to a symptomatic tumor. Our model supports a critical occult transition in tumor development during which time a transformed cell becomes a bona fide neoplasm. Lineage tracing and test-by-transplantation reveals that nonlinear progression during or prior to the occult transition gives rise to nascent lethal cancers at screen detection. Simulations illustrate how occult transition rates are a critical determinant of tumor growth and malignancy in the lifetime of a host. Our data provides direct experimental evidence that cancers can deviate from the predictable linear progression model foundational to current screening paradigms.

Dichotomous role of the serine/threonine kinase MAP4K4 in pancreatic ductal adenocarcinoma onset and metastasis through control of AKT and ERK pathways.

MAP4K4 is a serine/threonine kinase of the STE20 family involved in the regulation of actin cytoskeleton dynamics and cell motility. It has been proposed as a target of angiogenesis and inhibitors show potential in cardioprotection. MAP4K4 also mediates cell invasion in vitro, is overexpressed in various types of cancer, and is associated with poor patient prognosis. Recently, MAP4K4 has been shown to be overexpressed in pancreatic cancer, but its role in tumour initiation, progression, and metastasis is unknown. Here, using the KrasG12D Trp53R172H Pdx1-Cre (KPC) mouse model of pancreatic ductal adenocarcinoma (PDAC), we show that deletion of Map4k4 drives tumour initiation and progression. Moreover, we report that the acceleration of tumour onset is also associated with an overactivation of ERK and AKT, two major downstream effectors of KRAS, in vitro and in vivo. In contrast to the accelerated tumour onset caused by loss of MAP4K4, we observed a reduction in metastatic burden with both the KPC model and in an intraperitoneal transplant assay indicating a major role of MAP4K4 in metastatic seeding. In summary, our study sheds light on the dichotomous role of MAP4K4 in the initiation of PDAC onset, progression, and metastatic dissemination. It also identifies MAP4K4 as a possible druggable target against pancreatic cancer spread, but with the caveat that targeting MAP4K4 might accelerate early tumorigenesis. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Abstract PR014: Cytokine mediated epigenetic reprogramming of CRC primary tumors drives liver metastasis

Abstract An emerging hypothesis is that metastatic potential is determined largely by the combination of genetic alterations that initiate the primary tumor and subsequent transcriptional reprogramming through epigenetic mechanisms. Here we leverage integrative, multi-omic profiling of primary and metastatic colorectal cancer tumors in combination with functional screens and disease models to understand drivers of liver metastasis. Through performing bulk and single cell RNA-sequencing, whole exome sequencing, and H3K27Ac ChIP-seq of primary tumors and metastatic lesions in the liver, we identified dramatic enhancer remodeling in metastatic disease. In specific, we identified a region on chromosome 20 upstream of the signaling protein SIRPA containing 7 differentially active enhancers in metastatic disease. Not only do the enhancers show an in silico enrichment for TCF7 binding sites but we showed them in vitro to be reliant upon TCF7 through the use of a reporter system. To confirm the regulatory potential of TCF7 on SIRPA we performed CRISPRa mediated overexpression of TCF7. This resulted in significant increase in SIRPA levels. We then showed each gene to be functional drivers of metastasis and migration with in vitro and in vivo models. Through CRISPR mediated KO and CRISPRa mediated overexpression, we showed both genes to be necessary and sufficient for migration in a wound healing assay and invasion in a transwell assay. When the lines were transplanted orthotopically to the cecum, we saw a reduction in metastatic burden in the liver with the loss of either gene. To identify liver microenvironmental factors that lead to the upregulation of TCF7 and subsequent activity of SIRPA we assayed conditioned media derived from immortalized hepatocytes. Through the use of a cytokine array, we have shown the hepatocyte produced CCL2 is able to induce TCF7 and subsequently SIRPA in a panel of CRC cell lines and organoids. Importantly we showed the continued production of CCL2 is important for the survival of the cancer cell in the liver microenvironment through both genetic and drug intervention experiments. Together this data indicates an important signaling node in colorectal cancer metastasis to the liver. Here we propose a model in which secreted liver factors, such as CCL2, reprogram genes within the tumor cell to promote growth and survival of the liver. These genes, SIRPA, and TCF7, represent potential therapeutic targets. Targeting these proteins may kill existing metastatic lesions as well as block new metastases from forming which will be important in improving the outcomes of patients with oligometastatic and premetastatic disease. Citation Format: Jonathan Rennhack, Arika Dwivedi, Nathan Wu, Brian Shim, Alba Font-Tello, Nikolas Kesten, Andrew Aguirre, Henry Long, Kimmie Ng, William C. Hahn. Cytokine mediated epigenetic reprogramming of CRC primary tumors drives liver metastasis [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 PR014.

A randomised, double-blind, dose-finding, phase II multicentre study of ODX in the treatment of patients with castration-resistant prostate cancer and skeletal metastases

AimsThis study aimed to assess the efficacy and safety of ODX, a novel, cytotoxic, bone-targeting drug candidate, in castration-resistant prostate cancer bone metastatic disease. MethodsPatients with progressive disease were randomised to ten cycles of ODX, intravenous infusion Q2W (3, 6, and 9 mg/kg, respectively). The primary objective was to assess the relative change from baseline in bone alkaline phosphatase (B-ALP) and serum-aminoterminal-propeptide of Type I procollagen (S-P1NP) at 12 weeks. The inclusion criteria selected were broad, and a double-blind design was used to ensure objective recruitment of patients for the assessment of efficacy. None of the patients received bone-protecting agents during the ODX treatment period. ResultsFifty-five 21,20 and 14) patients were randomised to ODX (3, 6 and 9 mg/kg), respectively. The lower number of patients in arm 3 was due to too low a recruitment rate towards the end of the study. The median treatment time were 14, 13 and 14 weeks, respectively. The decrease in B-ALP at 12 weeks in study arms 3, 6 and 9 mg/kg was seen in 6/15 (40%), 8/12 (67%) and 5/12 (42%) patients, respectively, whereas the corresponding numbers for P1NP were 8/15 (53%), 8/12 (67%), and 4/12 (33%), respectively. The median decrease in B-ALP and P1NP at 12 weeks for study arms 3, 6 and 9 mg/kg were 37%, 14% and 43%, respectively, and 51%, 40% and 64%, respectively. The decrease in serum C-terminal telopeptide at 12 weeks was seen in the vast majority of patients and in about one-third of patients in bone scan index. ODX was well tolerated, and no drug-related serious adverse events occurred. There were no significant differences between study arms regarding efficacy and safety. ConclusionsODX was well tolerated and demonstrated inhibitory effects on markers related to the vicious cycle in bone at all three doses. The reduction in metastatic burden, assessed with bone scan index, supports this finding. Studies with continued ODX treatment until disease progression are being planned (ClinicalTrials.gov Identifier: NCT02825628).

Abstract 1785: Eribulin alters the chromatin landscape to induce MET, attenuating metastatic progression sensitizing breast tumors to subsequent chemotherapy

Abstract Introduction: Two major hurdles faced in clinical cancer therapy are metastatic progression that results in the majority of cancer deaths, and resistance to therapy that fuels tumor relapse. The epithelial-mesenchymal transition (EMT) is a reversible cellular program that contributes to the intratumoral heterogeneity of carcinoma cells and confers traits that promote metastatic progression and resistance to chemotherapy. The standard of care for triple negative breast cancer (TNBC) remains neoadjuvant chemotherapy where cyclophosphamide, doxorubicin and paclitaxel are administered in combination. In case of advanced/metastatic disease, third-line eribulin is administered as a single agent. In some cases of advanced disease, vinorelbine has also demonstrated efficacy. Eribulin, vinorelbine, and paclitaxel are microtubule inhibitors that act via mitotic blockade. Knowledge gap: Despite major advances in our understanding, the contributions of EMT research to improvements in cancer therapy have been minimal and yielded no clinically viable strategies to selectively modulate EMT in human tumors. Results: Our new data reveals that eribulin, in addition to blocking tubulin polymerization, utilizes epigenetic mechanisms to induce MET. Using thermal proteome profiling and mass spectrometry analyses, we identify candidate target proteins of eribulin that could enable it to act in an epigenetic fashion to induce MET.Treating TNBC patient-derived xenograft tumors with eribulin induces a significant reduction in metastatic burden, with the remaining metastatic colonies exhibiting a well-differentiated histopathology. Eribulin efficiently induce MET in cells that have received no prior chemotherapy; in cells pretreated with anthracyclines and taxanes, eribulin is less efficient at MET induction. Additionally, cells that were resistant to first-line taxane treatment were also resistant to eribulin-induced cell death. Using patient-derived xenograft (PDX) models, we have determined the optimal sequence of eribulin administration to maximize its MET-inducing and cytotoxic effects. Conclusions: We uncover that, working through altering the chromatin landscape and transcriptional profile of cells, eribulin induces MET and primes cells for subsequent chemotherapy. Additionally, the activity of eribulin is highest when administered in the treatment-naïve setting. By developing an understanding of eribulin’s mechanism of action, treatment strategies can be optimized to maximize therapeutic potential by exploiting its effects in both mitotic blockade as well as MET. Upon completion of this study, we will better understand how MET-induced tumor differentiation would work and the impact that it would have on current conventional therapeutic strategies. Acknowledgements: Eribulin studies funded by Eisai Inc. Citation Format: Meisam Bagheri, Nevena B. Ognjenovic, Gadisti Aisha Mohamed, Ian S. LaCroix, Andrew McCray, Kristen E. Muller, Xiaofeng Wang, Scott A. Gerber, Diwakar R. Pattabiraman. Eribulin alters the chromatin landscape to induce MET, attenuating metastatic progression sensitizing breast tumors to subsequent chemotherapy [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 1785.

Abstract P3-06-01: Eribulin alters the chromatin landscape to induce MET, attenuating metastatic progression and sensitizing breast tumors to subsequent chemotherapy

Abstract Introduction: Two major hurdles faced in clinical cancer therapy are metastatic progression that results in the majority of cancer deaths, and resistance to therapy that fuels tumor relapse. The epithelial-mesenchymal transition (EMT) is a reversible cellular program that contributes to the intratumoral heterogeneity of carcinoma cells and confers traits that promote metastatic progression and resistance to chemotherapy. The standard of care for triple negative breast cancer (TNBC) remains neoadjuvant chemotherapy where cyclophosphamide, doxorubicin and paclitaxel are administered in combination. In case of advanced/metastatic disease, third-line eribulin is administered as a single agent. In some cases of advanced disease, vinorelbine has also demonstrated efficacy. Eribulin, vinorelbine, and paclitaxel are microtubule inhibitors that act via mitotic blockade. Knowledge gap: Despite major advances in our understanding, the contributions of EMT research to improvements in cancer therapy have been minimal and yielded no clinically viable strategies to selectively modulate EMT in human tumors. Results: Our new data reveals that eribulin, in addition to blocking tubulin polymerization, utilizes microtubule-independent mechanism to epigenetically induce MET. Using thermal proteome profiling and mass spectrometry analyses, we identify candidate target proteins of eribulin that enable it to act in an epigenetic fashion to induce MET.Treating TNBC patient-derived xenograft tumors with eribulin induces a significant reduction in metastatic burden, with the remaining metastatic colonies exhibiting a well-differentiated histopathology. Eribulin efficiently induce MET in cells that have received no prior chemotherapy; in cells pretreated with anthracyclines and taxanes, eribulin is less efficient at MET induction. Additionally, cells that were resistant to first-line taxane treatment were also resistant to eribulin-induced cell death. Using patient-derived xenograft (PDX) models, we have determined the optimal sequence of eribulin administration to maximize its MET-inducing and cytotoxic effects. Conclusions: We uncover that, working through altering the chromatin landscape and transcriptional profile of cells, eribulin induces MET and primes cells for subsequent chemotherapy. Additionally, the efficacy of eribulin is highest when administered in the treatment-naïve setting. By developing an understanding of eribulin’s mechanism of action, treatment strategies can be optimized to maximize therapeutic potential by exploiting its effects in both mitotic blockade as well as MET. Upon completion of this study, we will better understand how MET-induced tumor differentiation would work and the impact that it would have on current conventional therapeutic strategies. Acknowledgements: Eribulin studies funded by Eisai Inc. Citation Format: Diwakar R Pattabiraman, Meisam Bagheri, Nevena B Ognjenovic, Ian S LaCroix, Scott A Gerber. Eribulin alters the chromatin landscape to induce MET, attenuating metastatic progression and sensitizing breast tumors to subsequent chemotherapy [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-06-01.

Doxorubicin Conjugation to Reovirus Improves Oncolytic Efficacy in Triple-Negative Breast Cancer

Breast cancer is the second leading cause of cancer-related deaths in women in the United States. The triple-negative breast cancer (TNBC) subtype associates with higher rates of relapse, shorter overall survival, and aggressive metastatic disease. Hormone therapy is ineffective against TNBC, leaving patients with limited therapeutic options. Mammalian orthoreovirus (reovirus) preferentially infects and kills transformed cells, and a genetically engineered reassortant reovirus infects and kills TNBC cells more efficiently than prototypical strains. Reovirus oncolytic efficacy is further augmented by combination with topoisomerase inhibitors, including the frontline chemotherapeutic doxorubicin. However, long-term doxorubicin use correlates with toxicity to healthy tissues. Here, we conjugated doxorubicin to reovirus (reo-dox) to control drug delivery and enhance reovirus-mediated oncolysis. Our data indicate that conjugation does not impair viral biology and enhances reovirus oncolytic capacity in TNBC cells. Reo-dox infection promotes innate immune activation, and crosslinked doxorubicin retains DNA-damaging properties within infected cells. Importantly, reovirus and reo-dox significantly reduce primary TNBC tumor burden in vivo, with greater reduction in metastatic burden after reo-dox inoculation. Together, these data demonstrate that crosslinking chemotherapeutic agents to oncolytic viruses facilitates functional drug delivery to cells targeted by the virus, making it a viable approach for combination therapy against TNBC.

VCAM-1 targeted alpha-particle therapy for early brain metastases.

BackgroundBrain metastases (BM) develop frequently in patients with breast cancer. Despite the use of external beam radiotherapy (EBRT), the average overall survival is short (6 months from diagnosis). The therapeutic challenge is to deliver molecularly targeted therapy at an early stage when relatively few metastatic tumor cells have invaded the brain. Vascular cell adhesion molecule 1 (VCAM-1), overexpressed by nearby endothelial cells during the early stages of BM development, is a promising target. The aim of this study was to investigate the therapeutic value of targeted alpha-particle radiotherapy, combining lead-212 (212Pb) with an anti–VCAM-1 antibody (212Pb-αVCAM-1).MethodsHuman breast carcinoma cells that metastasize to the brain, MDA-231-Br-GFP, were injected into the left cardiac ventricle of nude mice. Twenty-one days after injection, 212Pb-αVCAM-1 uptake in early BM was determined in a biodistribution study and systemic/brain toxicity was evaluated. Therapeutic efficacy was assessed using MR imaging and histology. Overall survival after 212Pb-αVCAM-1 treatment was compared with that observed after standard EBRT.Results 212Pb-αVCAM-1 was taken up into early BM with a tumor/healthy brain dose deposition ratio of 6 (5.52e108 and 0.92e108) disintegrations per gram of BM and healthy tissue, respectively. MRI analyses showed a statistically significant reduction in metastatic burden after 212Pb-αVCAM-1 treatment compared with EBRT (P < 0.001), translating to an increase in overall survival of 29% at 40 days post prescription (P < 0.01). No major toxicity was observed.ConclusionsThe present investigation demonstrates that 212Pb-αVCAM-1 specifically accumulates at sites of early BM causing tumor growth inhibition.

A radiobiological model of metastatic burden reduction for molecular radiotherapy: application to patients with bone metastases

Skeletal tumour burden is a biomarker of prognosis and survival in cancer patients. This study proposes a novel method based on the linear quadratic model to predict the reduction in metastatic tumour burden as a function of the absorbed doses delivered from molecular radiotherapy treatments.The range of absorbed doses necessary to eradicate all the bone lesions and to reduce the metastatic burden was investigated in a cohort of 22 patients with bone metastases from castration-resistant prostate cancer. A metastatic burden reduction curve was generated for each patient, which predicts the reduction in metastatic burden as a function of the patient mean absorbed dose, defined as the mean of all the lesion absorbed doses in any given patient. In the patient cohort studied, the median of the patient mean absorbed dose predicted to reduce the metastatic burden by 50% was 89 Gy (interquartile range: 83–105 Gy), whilst a median of 183 Gy (interquartile range: 107–247 Gy) was found necessary to eradicate all metastases in a given patient. The absorbed dose required to eradicate all the lesions was strongly correlated with the variability of the absorbed doses delivered to multiple lesions in a given patient (r = 0.98, P < 0.0001). The metastatic burden reduction curves showed a potential large reduction in metastatic burden for a small increase in absorbed dose in 91% of patients.The results indicate the range of absorbed doses required to potentially obtain a significant survival benefit. The metastatic burden reduction method provides a simple tool that could be used in routine clinical practice for patient selection and to indicate the required administered activity to achieve a predicted patient mean absorbed dose and reduction in metastatic tumour burden.

Abstract B49: Sustained IFNalpha treatment inhibits metastasis in 4T1 mammary carcinoma and correlates with decreasing myeloid-derived suppressor cells

Abstract In order to model pharmacokinetics and pharmacodynamics of pegylated IFNalpha, we utilized hydrodynamic delivery technology using minicircle DNA vectors encoding universal IFNalpha as a rapid and cost-effective method to obtain sustained levels of serum IFNalpha2alpha1. This approach is particularly useful for delivering secreted molecules, such as cytokines, as transfected hepatocytes secrete the transgene product in a paracrine manner providing a serum exposure profile resembling that reported in PEG-intron-treated patients. We found that sustained exposure of IFNalpha2alpha1 induced inhibition of metastasis. Primary 4T1 tumor growth was partially inhibited, while significant decreases in pulmonary metastatic burden were consistently observed. Further evidence that the observed inhibition of metastasis was independent of potential effects on the primary tumor was tested by resecting the primary tumor prior to treatment, which did not alter the outcome. Importantly, sustained IFNalpha2alpha1 treatment correlated with a significant increase in the survival of tumor-bearing animals. IFNalpha effects are plieotropic resulting in potential activation of numerous immune cell types associated with both innate and adaptive immunity. However, the mechanisms associated with IFNalpha-induced inhibition of lung metastasis were largely engendered by innate immune cells since treatment in BALB/c mice deficient for T and B cells did not affect the capacity of IFNalpha to inhibit pulmonary metastasis when compared to wild-type animals. The importance of NK cells has recently been reported as an important innate component for IFNalpha induced reduction of metastatic burden. However, little is known about IFNalpha-regulation of other innate cell types that may contribute to the inhibition of tumor metastasis. Here we report IFNalpha2alpha1 treatment induced marked decreases in 4T1-induced expansion and activation of immature CD11b+ Gr-1+ myeloid cells (aka: MDSC), which have been reported to contribute to metastatic potential. These results correlated with similar decreases in metastatic burden in mice following chlodronate-liposome depletion of CD11b+ Gr-1+ myeloid cells. Citation Format: Christina Kochel, Kalyan Pande, Roanna Ueda, Dan Gorman, Drake Laface. Sustained IFNalpha treatment inhibits metastasis in 4T1 mammary carcinoma and correlates with decreasing myeloid-derived suppressor cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B49.

Determinants of metastatic competency in colorectal cancer.

Colorectal cancer (CRC) is one of the most common cancer types and represents a major therapeutic challenge. Although initial events in colorectal carcinogenesis are relatively well characterized and treatment for early‐stage disease has significantly improved over the last decades, the mechanisms underlying metastasis – the main cause of death – remain poorly understood. Correspondingly, no effective therapy is currently available for advanced or metastatic disease. There is increasing evidence that colorectal cancer is hierarchically organized and sustained by cancer stem cells, in concert with various stromal cell types. Here, we review the interplay between cancer stem cells and their microenvironment in promoting metastasis and discuss recent insights relating to both patient prognosis and novel targeted treatment strategies. A better understanding of these topics may aid the prevention or reduction of metastatic burden.

Emergent role of the fractalkine axis in dissemination of peritoneal metastasis from epithelial ovarian carcinoma.

Epithelial ovarian carcinoma is the most common cause of death from gynecologic cancers largely due to advanced, relapsed, and chemotherapy-resistant peritoneal metastasis, which is refractory to the currently used treatment approaches. Mechanisms supporting advanced and relapsed peritoneal metastasis are largely unknown, precluding development of more effective targeted therapies. In this study we investigated the function of a potentially targetable fractalkine axis in the formation and the development of advanced and relapsed peritoneal metastasis and its impact on patients’ outcomes. Our mouse model studies support a role for the fractalkine receptor (CX3CR1) in the initiation of peritoneal adhesion important for recolonization of relapsed peritoneal metastasis. We show that downregulation of CX3CR1 results in reduction of metastatic burden at several peritoneal sites commonly colonized by advanced and relapsed metastatic ovarian carcinoma. We show that the chemokine fractalkine (CX3CL1), an activating ligand of CX3CR1, regulates organ-specific peritoneal colonization. High expression of CX3CR1 correlates with significantly shorter survival, specifically in post-menopausal patients with advanced and terminal stages of the disease. Taken together, our studies support a key regulatory role for the fractalkine axis in advanced and relapsed peritoneal metastasis in epithelial ovarian carcinoma.

Abstract 4030: In-vivo evaluation of the anti-metastatic efficacy of small molecule Cathepsin L inhibitor KGP94 in a prostate cancer model

Abstract It is estimated that ∼85% of patients with advanced prostate cancer develop skeletal metastases; an occurrence that results in a substantial reduction in the quality of life and a drastic worsening of the prognosis. The development of novel therapeutic strategies that impair the metastatic process and associated skeletal adversities is therefore critical to improving prostate cancer patient survival. Tumor cells rely on proteolytic enzymes for successful establishment of metastases. Cathepsin L (CTSL) is a lysosomal cysteine protease that is up regulated in a wide range of human cancers including prostate cancer. The elevated secretion of CTSL by aggressively metastasizing tumor cells plays a key role in metastatic spread of tumor cells through proteolytic degradation of extracellular matrix and basement membrane components. In addition, tumor secreted cytokines have been shown to increase osteoclastic secretion of CTSL to promote bone resorption. Intervention strategies targeting CTSL may therefore serve a dual purpose by inhibiting both metastatic dissemination as well as bone resorption. The goal of present studies was to evaluate the anti-metastatic and anti-bone resorptive efficacy of CTSL inhibition using the small molecule CTSL inhibitor 3-bromophenyl-3-hydroxyphenyl-ketone thiosemicarbazone (KGP94). KGP94 treatment significantly impaired the metastatic phenotype of highly metastatic prostate cancer PC-3ML cells. Treatment with 25µM KGP94 decreased the migratory and invasive capacities of PC-3ML cells by 78 and 53% respectively. To validate the anti-metastatic efficacy of KGP94 in-vivo, luciferase labelled PC-3ML cells were injected into the left ventricle of male nu/nu mice followed by daily treatment with 20mg/kg KGP94. Metastatic progression was monitored by weekly bioluminescence imaging using firefly luciferase reporter system. KGP94 treatment led to a significant reduction in metastatic tumor burden (65% reduction) and a reduction in metastatic incidence. Histological assessment of bones further confirmed the reduction in metastatic burden upon KGP94 treatment. CTSL inhibition also led to a significant increase in overall survival of tumor bearing mice (p=0.01). In vitro assessment of anti-resorptive function of KGP94 was performed using osteoclastogenesis and pit formation assays. KGP94 treatment of RAW264.7 osteoclast precursor cells led to a significant decrease (72%) in osteoclast formation as demonstrated by a decrease in the number of TRAP+ multinucleate cells. KGP94 also inhibited the bone resorptive activity of osteoclasts as indicated by a significant reduction in the number of resorptive pits formed by osteoclasts on bone slices. In conclusion, our findings suggest that CTSL inhibition using small molecule KGP94 can significantly impair metastatic dissemination of prostate cancer cells and alleviate bone resorption. Citation Format: Dhivya R. Sudhan, Dietmar W. Siemann. In-vivo evaluation of the anti-metastatic efficacy of small molecule Cathepsin L inhibitor KGP94 in a prostate cancer model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4030. doi:10.1158/1538-7445.AM2014-4030

Abstract 3943: Cancer cell cooperation in the bone metastatic niche.

Abstract Advanced prostate cancer (PCa) frequently spreads to the skeleton and bone metastases are a major cause of morbidity and the main cause of death from this malignancy. The effective treatment of PCa disseminated to the skeleton remains an unmet clinical need. This lack of curative therapeutic approaches stems from our limited knowledge of the molecular interactions underpinning the colonization, survival and growth of cancer cells in the bone microenvironment. In this study, we focused on the autocrine and paracrine interactions involving different phenotypes of Disseminated Tumor Cells (DTCs) and the surrounding bone marrow stroma. These functional interactions are likely to play a crucial role in dictating the fate of DTCs and their disruption could interfere with the supportive role of the metastatic niche. By using comparative gene expression analysis we have demonstrated that IL-1β and CXCL6 determine the bone-metastatic behavior of human prostate cancer cells inoculated in the left cardiac ventricle of SCID mice. Since the tumor-producing cancer cells secrete both signaling molecules, we speculated that their roles include the establishment of cross talk with the surrounding cells of the bone stroma. The stromal cells would reciprocate with an increased or de novo production of trophic factors that promote metastatic progression. These local factors could also support DTC types that would normally be unable to initiate a metastatic niche, resulting in a form of cancer cell cooperation at distant sites. To test this hypothesis, we generated skeletal lesions in mice using Td-Tomato-labeled metastatic cells inoculated via the intracardiac route, followed by a second inoculation with GFP-labeled PCa cells lacking metastatic potential. We found that under these conditions the resulting skeletal lesions were composed of both cell types, indicating that metastasis-initiating cells could support subsequent colonization by cancer cells that normally fail to survive in the bone microenvironment. We then simultaneously inoculated animals with both cell types and discovered that the supportive influence of metastasis-initiating cells on non-metastatic cells is exerted immediately after homing to the skeleton. Further, we used Laser Capture Microdissection (LCM) to confirm high expression of IL-1β and CXCL6 in DTCs in metastatic lesions and then to identify signaling molecules and stromal cell types implicated in this novel cooperation mechanism in bone-metastatic lesions. These data may help identify multiple drug targets that, if clinically exploited, could lead to a substantial reduction in metastatic burden and concomitant increase in quality of life in the advanced prostate cancer patient population. Citation Format: Qingxin Liu, Kristina Shahriari, Marina Sazanovich, Danielle Jernigan, Alessandro Fatatis. Cancer cell cooperation in the bone metastatic niche. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3943. doi:10.1158/1538-7445.AM2013-3943

Host-related carcinoembryonic antigen cell adhesion molecule 1 promotes metastasis of colorectal cancer

Liver metastasis is the predominant cause of colorectal cancer (CRC)-related mortality in developed countries. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a cell adhesion molecule with reduced expression in early phases of CRC development and thus functions as a tumor growth inhibitor. However, CEACAM1 is upregulated in metastatic colon cancer, suggesting a bimodal role in CRC progression. To investigate the role of this protein in the host metastatic environment, Ceacam1(-/-) mice were injected intrasplenically with metastatic MC38 mouse CRC cells. A significant reduction in metastatic burden was observed in Ceacam1(-/-) compared with wild-type (WT) livers. Intravital microscopy showed decreased early survival of MC38 cells in Ceacam1(-/-) endothelial environment. Metastatic cell proliferation within the Ceacam1(-/-) livers was also diminished. Bone marrow-derived cell recruitment, attenuation of immune infiltrates and diminished CCL2, CCL3 and CCL5 chemokine production participated in the reduced Ceacam1(-/-) metastatic phenotype. Transplantations of WT bone marrow (BM) into Ceacam1(-/-) mice fully rescued metastatic development, whereas Ceacam1(-/-) BM transfer into WT mice showed reduced metastatic burden. Chimeric immune cell profiling revealed diminished recruitment of CD11b(+)Gr1(+) myeloid-derived suppressor cells (MDSCs) to Ceacam1(-/-) metastatic livers and adoptive transfer of MDSCs confirmed the involvement of these immune cells in reduction of liver metastasis. CEACAM1 may represent a novel metastatic CRC target for treatment.

Abstract 2431: Inhibition of metastasis through therapeutic targeting of focal adhesion kinase (FAK) in pancreatic cancer

Abstract BACKGROUND: Cancer mortality is primarily caused by metastatic disease; it has therefore been suggested that the preclinical evaluation of novel therapies should include testing in metastatic models of disease. This is especially relevant in light of recent reports of both conventional and targeted therapies being associated with accelerated metastases despite maintenance of primary tumor control. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase which also acts as molecular scaffold, and localizes to focal adhesions, contact points between the cell and the extra-cellular matrix. Downstream of integrins and transmembrane receptors, FAK acts as a signaling hub, participating in processes integral to tumor progression including cell survival, proliferation, angiogenesis, migration and invasion. In this ongoing study, we explore the efficacy of selective genetic and pharmacologic FAK inhibition in preclinical models of human pancreatic cancer, including spontaneously metastasizing, cell-line derived and primary xenografts. METHODS &amp; RESULTS: We constructed modified pancreatic cancer cell lines using retroviral over-expression of the dominant negative FRNK (FAK related non-kinase). Pharmacologic inhibition of FAK was performed with PF-562271 (Pfizer), a selective FAK/Pyk2 inhibitor which recently completed Phase I testing. Adhesion, invasion and migration in vitro assays were conducted using the xCELLigence RTCA system. In vivo efficacy of FAK inhibition was evaluated in orthotopic xenograft models. In vitro studies demonstrated that both modalities of FAK inhibition resulted in impaired adhesion, invasion and migration and 3D colony formation, without comparable effects on viability, proliferation or colony formation in 2D. Expression of dominant-negative FRNK resulted in 49% smaller xenograft tumors (p=0.003), a 78% reduction in metastatic disease (p&amp;lt; 0.001) and longer overall survival (p &amp;lt; 0.001). There was a similar, 46% trend to reduction of metastatic burden (p=0.089) in orthotopic cell line xenograft models treated with PF-562271, despite a lack of significant primary tumor growth delay. CONCLUSIONS: Our data suggest a potential utility of selective FAK inhibition in delaying metastatic progression of pancreatic cancer. This is particularly relevant clinically since patients frequently present with locally advanced, unresectable disease and subsequently develop metastases. Pharmacologic FAK inhibition (in combination with conventional therapies) is currently being evaluated in neo/adjuvant models of this disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2431. doi:10.1158/1538-7445.AM2011-2431

Tumour burden and interleukin-2 dose affect the interaction between low-dose total body irradiation and interleukin 2

Low-dose total body irradiation (LTBI) has a synergistic immune-mediated antitumour effect when used in combination with interleukin 2 (IL-2) in a murine metastatic malignant melanoma model. To optimise the use of this combination treatment this study was performed to test the effect of tumour burden and dose of both LTBI and IL-2 on the therapeutic potential of this treatment strategy. Ten-week-old female C57BL/6 mice were inoculated intravenously (day 0) with 1 million B16F1 malignant melanoma cells. Groups of mice received no treatment, a single fraction of LTBI alone, IL-2 treatment alone, or a combination of LTBI and IL-2. Two doses of LTBI and IL-2 were tested. LTBI was given on day +10 and IL-2 treatment started on day +11. On day +18 the mice were killed. The lungs were removed and analysed for tumour burden. Lung sections were also tested for infiltrating leucocytes using immunohistochemical staining. In one experiment, mice were treated at day +7 with low-dose IL-2 with and without LTBI. LTBI (in the two tested doses) showed no independent therapeutic effects. An IL-2 dose of 300,000 Cetus units (CU) that was effective and showed synergism with LTBI when mice were treated on day +7 failed to show a therapeutic effect when mice were treated on day +10, at which time the initial tumour burden had doubled. High-dose IL-2 (600,000 CU), in contrast, led to a significant reduction in metastatic burden compared to the control group. Combining high-dose IL-2 with LTBI led to a further significant reduction in tumour burden. Moreover, this combination was associated with a less severe vascular leakage syndrome compared to IL-2 alone. IL-2 and combination treatment was associated with an increase in the number of tumour-infiltrating immune cells, but only the number of tumour-infiltrating natural killer cells reflected therapeutic efficacy. It was concluded that tumour burden at the time of treatment and IL-2 dose are two crucial factors affecting the synergism between LTBI and IL-2. The combination may not only be more effective than IL-2 alone but also less toxic.

In vivo videomicroscopy reveals differential effects of the vascular-targeting agent ZD6126 and the anti-angiogenic agent ZD6474 on vascular function in a liver metastasis model

Metastases require a functional blood supply for progressive growth. Thus, therapies that target metastatic vasculature have potential clinical utility. The effects of the vascular-targeting agent (VTA), ZD6126, and the anti-angiogenic agent, ZD6474, on vascular development and function within metastases were compared in an experimental liver metastasis model. Ras-transformed PAP2 fibroblasts were injected into the mesenteric veins of SCID mice to produce a control liver metastasis burden of approximately 40% at 14 days. Mice given a single dose of ZD6126 (200 mg/kg, i.p.) on day 13 were examined 24 h later. Histology revealed a significant reduction in metastatic burden, associated with extensive tumor necrosis, increased tumor cell apoptosis and a reduction in tumor-associated vasculature. In vivo videomicroscopy (IVVM) revealed disrupted, non-functional vascular channels within metastases, with no blood flow. Mice given ZD6474 on days 4 to 10 (50 mg/kg daily, oral gavage) were examined on day 11. Histology revealed a lower metastatic burden, significant reductions in metastasis size and vasculature, and a significant increase in tumor cell apoptosis. IVVM revealed extensive reductions in vascularity and blood flow within metastases. Neither ZD6126 nor ZD6474 treatment affected surrounding normal liver tissue. This study shows that both agents can reduce experimental liver metastasis with no apparent effect on normal vasculature. However, these reductions were attained through distinct effects on the metastatic vasculature. Understanding differences in the modes of action of VTAs and anti-angiogenic agents will be important in optimizing their clinical application and in developing appropriate combination strategies.

Co-inoculation of human and murine carcinoma cells induces reciprocal suppression of metastasis by both cell lines.

The interactions of two cell lines having different metastatic properties, and the subsequent effects on dissemination were investigated using the chicken embryo metastasis assay. The highly aggressive human epidermoid cell line HEp-3 was tested alone or mixed with the mouse colon carcinoma cell line CL26 in this assay. When inoculated individually, each cell line forms experimental metastases in the chicken embryo, but only the HEp-3 cells give rise to spontaneous metastases. In embryos co-inoculated with both cell lines there was an overall reduction in metastatic burden in both the spontaneous and experimental metastasis assays. Furthers studies revealed that CL26 cells, when co-inoculated with HEp-3 cells did not acquire the ability to spontaneously metastasize. However, in the presence of CL26 cells, spontaneous HEp-3 metastasis was reduced. Intravenous co-inoculation of HEp-3 and CL26 cells also resulted in a reciprocal suppression of experimental metastasis by both cell lines. These studies demonstrate that the interactions of adjacent, phenotypically different tumor cells can have a suppressive effect on dissemination of one or both cell types.