Cell migration is a key procedure involved in many biological processes including embryological development, tissue formation, immune defense or inflammation, and cancer progression. How physical, chemical, and molecular aspects can affect cell motility is a challenge to understand migratory cells behavior. In vitro assays are excellent approaches to extrapolate to in vivo situations and study live cells behavior. Here we present four in vitro protocols that describe step-by-step cell migration, invasion and adhesion strategies and their corresponding image data quantification. These current protocols are based on two-dimensional wound healing assays (comparing traditional pipette tip-scratch assay vs. culture insert assay), 2D individual cell-tracking experiments by live cell imaging and three-dimensional spreading and transwell assays. All together, they cover different phenotypes and hallmarks of cell motility and adhesion, providing orthogonal information that can be used either individually or collectively in many different experimental setups. These optimized protocols will facilitate physiological and cellular characterization of these processes, which may be used for fast screening of specific therapeutic cancer drugs for migratory function, novel strategies in cancer diagnosis, and for assaying new molecules involved in adhesion and invasion metastatic properties of cancer cells.

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In vitro Cell Migration, Invasion, and Adhesion Assays: From Cell Imaging to Data Analysis.

Colorectal cancer (CRC) is one of the most common causes of cancer deaths worldwide and the number of CRC patients is increasing progressively. Despite the improvement of the surgical techniques and chemotherapy, we have not completely overcome this disease yet due to the metastases. Therefore, understanding the mechanisms through which metastasis occurs is important for overcoming CRC. Normal host cells in the tumor microenvironment, such as macrophages and fibroblasts, have been reported to promote the growth of CRCs. Although neutrophils were originally considered to have defensive functions against tumor cells, it has been revealed that some populations of neutrophils, called as tumor-associated neutrophils (TANs), have tumor-supportive functions. The plasticity between tumor-suppressive and -supportive neutrophils are regulated by transforming growth factor (TGF)-β and Interferon-β signaling. Some studies have demonstrated that TANs promote the spread of cancer cells to distant organs. TANs contribute to the tumor invasion and angiogenesis through the production of matrix metalloproteinase-9 (MMP9), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) in the primary and metastatic sites. Neutrophils also promotes tumor cell dissemination by capturing circulating tumor cells using neutrophil extracellular traps and promote their migration to distant sites. The neutrophil-to-lymphocyte ratio is a well-defined predictive marker for CRC patients. In this review, we highlight the molecular signaling between TANs and CRC cells and the possibility of TANs as a potential target for cancer therapy.

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The Role of Tumor-Associated Neutrophils in Colorectal Cancer

Tumours reprogram host physiology, metabolism and immune responses during cancer progression. The release of soluble factors, exosomes and metabolites from tumours leads to systemic changes in distant organs, where cancer cells metastasize and grow. These tumour-derived circulating factors also profoundly impact tissues that are rarely inhabited by metastatic cancer cells such as skeletal muscle and adipose tissue. In fact, the majority of patients with metastatic cancer develop a debilitating muscle-wasting syndrome, known as cachexia, that is associated with decreased tolerance to antineoplastic therapy, poor prognosis and accelerated death, with no approved treatments. In this Perspective, we discuss the development of cachexia in the context of metastatic progression. We briefly discuss how circulating factors either directly or indirectly promote cachexia development and examine how signals from the metastatic process can trigger and amplify this process. Finally, we highlight promising therapeutic opportunities for targeting cachexia in the context of metastatic cancers.

Understanding cachexia in the context of metastatic progression.

Our understanding of cancer biology has increased substantially over the past 30 years. Despite this, and an increasing pharmaceutical company expenditure on research and development, the approval of novel oncology drugs during the past decade continues to be modest. In addition, the attrition of agents during clinical development remains high. This attrition can be attributed, at least in part, to the clinical development being underpinned by the demonstration of predictable efficacy in experimental models of human tumours. This review will focus on the range of models available for the discovery and development of anticancer drugs, from traditional subcutaneous injection of tumour cell lines to mice genetically engineered to spontaneously give rise to tumours. It will consider the best time to use the models, along with practical applications and shortcomings. Finally, and most importantly, it will describe how these models reflect the underlying cancer biology and how well they predict efficacy in the clinic. Developing a line of sight to the clinic early in a drug discovery project provides clear benefit, as it helps to guide the selection of appropriate preclinical models and facilitates the investigation of relevant biomarkers.

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The role of mouse tumour models in the discovery and development of anticancer drugs.

Reversing cold tumors to hot: An immunoadjuvant-functionalized metal-organic framework for multimodal imaging-guided synergistic photo-immunotherapy.

Most cancer-related deaths are a result of metastasis, and thus the importance of this process as a target of therapy cannot be understated. By asking ‘how can we effectively treat cancer?’, we do not capture the complexity of a disease encompassing >200 different cancer types — many consisting of multiple subtypes — with considerable intratumoural heterogeneity, which can result in variable responses to a specific therapy. Moreover, we have much less information on the pathophysiological characteristics of metastases than is available for the primary tumour. Most disseminated tumour cells that arrive in distant tissues, surrounded by unfamiliar cells and a foreign microenvironment, are likely to die; however, those that survive can generate metastatic tumours with a markedly different biology from that of the primary tumour. To treat metastasis effectively, we must inhibit fundamental metastatic processes and develop specific preclinical and clinical strategies that do not rely on primary tumour responses. To address this crucial issue, Cancer Research UK and Cancer Therapeutics CRC Australia formed a Metastasis Working Group with representatives from not-for-profit, academic, government, industry and regulatory bodies in order to develop recommendations on how to tackle the challenges associated with treating (micro)metastatic disease. Herein, we describe the challenges identified as well as the proposed approaches for discovering and developing anticancer agents designed specifically to prevent or delay the metastatic outgrowth of cancer.

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A framework for the development of effective anti-metastatic agents.


 Background: Focal Adhesion Kinase (FAK) plays a key role in tumor cell growth, particularly immunosuppression, cancer cell invasion and metastasis and also contributes to multiple mechanisms underlying fibrosis. AMP945 is a selective oral inhibitor of FAK. Following a favorable pre-clinical evaluation, we report the results of a Phase 1, randomized, double-blinded, placebo-controlled single ascending dose (SAD) and multiple ascending dose (MAD) trial of AMP945.
 Methods: For the SAD portion, four cohorts of eight healthy volunteers aged between 18 and 56 years received either single doses of AMP945 (n=6/cohort) of 15, 30, 60, or 125 mg per oral or matching placebo (n=2/cohort) per oral. For the MAD portion, three cohorts of eight healthy volunteers received either AMP945 (n=6/cohort) at 25, 50, or 100 mg once daily (QD) per oral or matching placebo (n=2/cohort) per oral QD on each of 7 consecutive days. Blood plasma samples were collected for pharmacokinetic (PK) analyses pre-dose at 0.25, 0.5, 1, 2, 4, 6, 8, 12, 18, 24, 36, and 48 h post-dose in SAD cohorts, and at the same time points up to 24 h post-dose on Days 1 and 7 in the MAD cohorts. Target engagement through FAK activity (phosphorylation; p-FAK) was assessed in skin biopsies taken before and after dosing in the 125 mg AMP945 (SAD) and 25, 50, 100 mg AMP945 (MAD) cohorts. Safety and tolerability were assessed according to incidence, nature and severity of adverse events (AEs).
 Results: AMP945 was well tolerated at all doses studied. There was no increase in the incidence or severity of AEs with increasing doses and no treatment-related changes in laboratory parameters. AEs were mainly mild and deemed not related or unlikely related to AMP945 by the Investigator with headache, backpain and pyrexia being the most commonly observed events. After single doses of AMP945 the mean time to maximum plasma concentration ranged from 1 to 6 h post-dose. Maximum plasma concentration and area under the curve increased proportionally with dose. Mean Cmax values ranged from 55.4 to 407 ng/mL and AUC0-inf ranged from 1,086 to 10,567 h.ng/mL respectively. The mean half-life of AMP945 ranged from 15.7 to 23 h supporting the feasibility of QD dosing. The mean apparent volume of distribution ranged from 328 to 463 L, indicating wide tissue distribution. In skin biopsies, p-FAK levels following doses of AMP945 were reduced in a dose-dependent manner: there was a significant (linear) relationship observed between the decrease in p-FAK from baseline and AMP945 AUC0-inf following dosing with 25, 50, 100 and 125 mg of AMP945.
 Conclusions: AMP945, a selective FAK inhibitor, was well tolerated across SAD (15 to 125 mg) and MAD (25 to 100 mg) cohorts in healthy volunteers. AMP945 PK and pharmacodynamic data demonstrate wide tissue distribution and target engagement. These data support continued development of AMP945 in patients with solid tumors and fibrotic diseases in which FAK inhibition could be beneficial.
 Citation Format: John Lambert, Christopher J. Burns, Mark Devlin, Nicole Kruger, Jason Lickliter, Mark Sullivan, Warwick Tong. A phase 1 trial of AMP945, a potent and selective focal adhesion kinase inhibitor, in healthy volunteers [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 CT511.

Warwick Tong

Papers

A framework for the development of effective anti-metastatic agents.

Abstract CT511: A phase 1 trial of AMP945, a potent and selective focal adhesion kinase inhibitor, in healthy volunteers