Abstract
Cellular and molecular heterogeneity within tumors has long been associated with the progression of cancer to an aggressive phenotype and a poor prognosis. However, how such intratumoral heterogeneity contributes to the invasiveness of cancer is largely unknown. Here, using a tumor bioengineering approach, we investigate the interaction between molecular subtypes within bladder microtumors and the corresponding effects on their invasiveness. Our results reveal heterogeneous microtumors formed by multiple molecular subtypes possess enhanced invasiveness compared to individual cells, even when both cells are not invasive individually. To examine the molecular mechanism of intratumoral heterogeneity mediated invasiveness, live single cell biosensing, RNA interference, and CRISPR-Cas9 gene editing approaches were applied to investigate and control the composition of the microtumors. An agent-based computational model was also developed to evaluate the influence of NOTCH1 variation on DLL4 expression within a microtumor. The data indicate that intratumoral variation in NOTCH1 expression can lead to upregulation of DLL4 expression within the microtumor and enhancement of microtumor invasiveness. Overall, our results reveal a novel mechanism of heterogeneity mediated invasiveness through intratumoral variation of gene expression.
Highlights
In the United States, bladder cancer is a common malignancy, with an estimated 83,730 people diagnosed and over 17,200 individuals dying annually [1]
Using established cell lines with luminal and basal/squamous signatures, transient knockdown by siRNA, CRISPR knockout, and in silico models, we examine the influence of heterogeneity of NOTCH1-DLL4 signaling on bladder cancer invasion
This study demonstrated a combination of tumor bioengineering, single cell analysis, and computational modeling for investigating the influence of intratumoral heterogeneity on cancer invasiveness
Summary
In the United States, bladder cancer is a common malignancy, with an estimated 83,730 people diagnosed and over 17,200 individuals dying annually [1]. At the individual patient level, several groups have independently completed molecular characterization of human bladder cancer [2,3,4] These efforts led to the discovery of luminal, basal/squamous, and other transcriptional subtypes (stroma-rich and neuroendocrine-like) of the muscle-invasive bladder cancer [5]. Basal/squamous cancer cells are typically classified by elevated expression of specific cytokeratins, such as KRT5 and KRT14 [6] While it appears that basal/squamous tumors respond favorably to neoadjuvant, platinum-based chemo-therapy and targeted therapeutic approaches, recent reports indicate patients with basal/squamous bladder cancer have inferior overall and disease-specific survival rates [3]. Using established cell lines with luminal and basal/squamous signatures, transient knockdown by siRNA, CRISPR knockout, and in silico models, we examine the influence of heterogeneity of NOTCH1-DLL4 signaling on bladder cancer invasion. The plate was covered and returned to the cell culture incubator for 3 days before imaging
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