Cellular heterogeneity profiling by hyaluronan probes reveals an invasive but slow-growing breast tumor subset

Abstract

Tumor heterogeneity confounds cancer diagnosis and the outcome of therapy, necessitating analysis of tumor cell subsets within the tumor mass. Elevated expression of hyaluronan (HA) and HA receptors, receptor for HA-mediated motility (RHAMM)/HA-mediated motility receptor and cluster designation 44 (CD44), in breast tumors correlates with poor outcome. We hypothesized that a probe for detecting HA-HA receptor interactions may reveal breast cancer (BCa) cell heterogeneity relevant to tumor progression. A fluorescent HA (F-HA) probe containing a mixture of polymer sizes typical of tumor microenvironments (10-480 kDa), multiplexed profiling, and flow cytometry were used to monitor HA binding to BCa cell lines of different molecular subtypes. Formulae were developed to quantify binding heterogeneity and to measure invasion in vivo. Two subsets exhibiting differential binding (HA −/low vs. HA high ) were isolated and characterized for morphology, growth, and invasion in culture and as xenografts in vivo. F-HA-binding amounts and degree of heterogeneity varied with BCa subtype, were highest in the malignant basal-like cell lines, and decreased upon reversion to a nonmalignant phenotype. Binding amounts correlated with CD44 and RHAMM displayed but binding heterogeneity appeared to arise from a differential ability of HA receptor-positive subpopulations to interact with F-HA. HA high subpopulations exhibited significantly higher local invasion and lung micrometastases but, unexpectedly, lower proliferation than either unsorted parental cells or the HA −/low subpopulation. Querying F-HA binding to aggressive tumor cells reveals a previously undetected form of heterogeneity that predicts invasive/metastatic behavior and that may aid both early identification of cancer patients susceptible to metastasis, and detection/therapy of invasive BCa subpopulations.tumor cell heterogeneity | hyaluronan binding | heterogeneity index B reast tumors display substantial heterogeneity driven by genetic and epigenetic mechanisms (1-3). These processes select and support tumor cell subpopulations with distinct phenotypes in proliferation, metastatic/invasive proclivity, and treatment susceptibility that contribute to clinical outcomes. Currently, there is a paucity of biomarkers to identify these subpopulations (3-12). Although detection of genetic heterogeneity may itself be a breast cancer (BCa) prognostic marker (3, 13-15), the phenotypes manifested from this diversity are context-dependent. Therefore, phenotypic markers provide additional powerful tools for biological information required to design diagnostics and therapeutics. Glycomic approaches have enormous potential for revealing tumor cell phenotypic heterogeneity because glycans are themselves highly heterogeneous and their complexity reflects the nutritional, microenvironmental, and genetic dynamics of the tumors (16-18).We used hyaluronan (HA) as a model carbohydrate ligand for probing heterogeneity in glycosaminoglycan-BCa cell receptor interactions. We reasoned this approach...

Hyaluronan as a therapeutic target in human diseases

“…HA-mediated invasive cell motility is regulated by TGF-β [156, 157] and Src activation [157]. Furthermore, cellular heterogeneity profiling by HA probes reveals an invasive but slow-growing breast tumor subset [158]. We showed that fibroblasts isolated from transgenic mice overexpressing HAS2 had a greater capacity to invade matrix.…”

“…The peptide was also able to prevent nuclear accumulation of HA [158] and to inhibit prostate tumor cell migration toward to fragmented HA [304]. …”

Carcinoma Cell Hyaluronan as a “Portable” Cancerized Prometastatic Microenvironment

“…For example, they can promote the adhesion of tumor cells to microvessel endothelium as has been demonstrated for metastatic prostate carcinoma cells in culture [9]. Furthermore, breast tumor cell subpopulations that bind high levels of HA adhere to vessels and extravasate ex ovo more rapidly than tumor cells that bind little or no HA [16]. Pericellular HA matrices may participate in the conditioning of foreign microenvironments by stimulating the release of microvesicles from HAS and HA-rich cell microvilli [17].…”

Capturing cancer’s complexity

“…"We reveal many details that we're missing with lesssensitive methods." Veiseh recently used a fluorescent probe technique to detect and quantify the build-up of the sugar hyaluronan in a small percentage of the cells in slow-growing but invasive breast tumor cell lines that were thought to be homogenous; the results were reported last April (6). Eva Turley of the London Health Science Centre had previously linked the accumulation of hyaluronan to the likelihood that a tumor would spread (7).…”