Abstract
Simple SummaryThe main problem in treating advanced cancers is a metastatic spread when individual cancer cells leave the primary tumor and colonize to distant organs. In drug development, it is important to quantitatively assess effects of novel drug candidates on both primary tumors and metastasis. Unfortunately, current methods of monitoring metastasis in mouse models have low sensitivity and are not quantitative. Here, we developed a methodology to monitor drug effects on metastasis that is quantitative and has a very high sensitivity and resolution. In fact, it allows monitoring effects of drugs on individual cancer cells in animals.Imaging in monitoring metastasis in mouse models has low sensitivity and is not quantitative. Cell DNA barcoding, demonstrating high sensitivity and resolution, allows monitoring effects of drugs on the number of tumor and metastatic clones. However, this technology is not suitable for comparison of sizes of metastatic clones in different animals, for example, drug treated and untreated, due to high biological and technical variability upon tumor and metastatic growth and isolation of barcodes from tissue DNA. However, both numbers of clones and their sizes are critical parameters for analysis of drug effects. Here we developed a modification of the barcoding approach for monitoring drug effects on tumors and metastasis that is quantitative, highly sensitive and highly reproducible. This novel cell double-barcoding system allows simultaneously following the fate of two or more cell variants or cell lines in xenograft models in vivo, and also following the fates of individual clones within each of these populations. This system allows comparing effects of drugs on different cell populations and thus normalizing drug effects by drug-resistant lines, which corrects for both biological and technical variabilities and significantly increases the reproducibility of results. Using this barcoding system, we uncovered that effects of a novel DYRK1B kinase inhibitor FX9847 on primary tumors and metastasis is clone-dependent, while a distinct drug osimertinib demonstrated clone-independent effects on cancer cell populations. Overall, a cell double-barcoding approach can significantly enrich our understanding of drug effects in basic research and preclinical studies.
Highlights
Quantitative evaluation of drug effects on tumor growth and metastasis in xenograft animal models is critical for basic understanding of tumor dynamics and for anti-cancer drug development
The common approach to test for drug effects in preclinical studies is subcutaneous cancer cell injection, which is routinely used by CROs and is accepted by Food and Drug Administration (FDA)
Used approaches that involve imaging or direct measurements suffer from relatively low sensitivity and low resolution, leading to the inability to quantitatively evaluate the number of metastases and the sizes of individual metastasis
Summary
Quantitative evaluation of drug effects on tumor growth and metastasis in xenograft animal models is critical for basic understanding of tumor dynamics and for anti-cancer drug development. In many cases primary xenograft tumors formed by human cancer cells injected subcutaneously in nude mice do not generate detectable metastasis. To obtain such metastasis, injection of cells into orthotopic organs or the tail vein is done. Injection of cells into orthotopic organs or the tail vein is done These approaches, are elaborate and CROs rarely use such methods in the preclinical studies, especially since the U.S Food and Drug Administration (FDA) accepts preclinical data using subcutaneous xenografts (see, for example, [1–3]). When metastasis develops in animals, detection by imaging reflects a combination of the number of metastases and their individual sizes without distinguishing these parameters, which is very important
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