Blood-derived non-extracellular vesicle proteins as potential biomarkers for the diagnosis of early ER+ breast cancer and detection of lymph node involvement

Extracellular vesicles (EVs) are membrane surrounded structures released by different cell types that are involved in cellular communication and are emerging as potential therapeutic and diagnostic targets in cancer¹ as in the case of early pancreatic cancer².

EVs can be classified in several subtypes based on their size, shape, and supposed origin. Exosomes are defined as ∼30–100 nm vesicles which originate from multivesicular bodies (MVB) and contain late endosomal markers^3,4, although biochemically indistinguishable vesicles can bud directly from the plasma membrane^3,5. Microvesicles or shedding vesicles are generally larger (>200 nm), are more variable in shape and density, and likely originate from the plasma membrane^4,6,7. EVs may contain proteins, lipids, and RNAs, however how these components are sorted into EVs remains unclear.

Tumor-derived EVs are also critical components for preparing the tumor microenvironment because they enable tumor cells to escape from the immunological surveillance⁸ and help in the setting of a pre-metastatic niche for the engraftment of detached cancer cells⁹. Both exosomes and MVs have been extensively studied and attributed various important physiological roles in cancer^10,11. For instance, EVs have been found to play an important role in every phase of cancer development from cancer initiation, invasion and metastasis¹². For these reasons, EVs are potential therapeutic and diagnostic targets in cancer and EV-derived biomarkers maybe useful for predicting future metastatic development and identify metastasis sites¹³.

ER+ (estrogen receptor positive) breast cancer (BC) represents 60–80% of all BC cases^14,15. Here we describe our preliminary findings exploring the role of tumour derived EVs biomarkers that could ultimately be used as part of a test kit for the detection of early ER+ BC and lymph node involvement.

Methods

Results and discussion

Clinical data on the EVs isolated from BC patient’s plasma samples and cell lines can be found in Table 1. The method used for EV isolation also precipitates lipoproteins and immunocomplexes (IC) which are known possible contaminants¹⁹. However, samples submitted for mass spectrometry analysis showed none of the recognised contaminants of high speed centrifugation. In the two patients with early BC (Table 2a), we detected HCG1745306 isoform CRA-a, a protein from the family of alpha type haemoglobins and for the patient with lymph node involvement, we detected histone H1.2 (Table 2 a–b). HCG1745306 isoform CRA-a was only present in the two patients with early BC with Mascot scores of 3208.8 and 3966.5, respectively and absent in all controls and other patient samples.

Histone H1.2 was also detected in samples from the two patients with bone metastases, a parental primary BC sample and metastatic SKBr-3, MDA468, MDA231 cell lines. However, histone H1.2 was absent from the plasma sample of a patient with multiple metastases, from the non-metastatic MCF7 cell line (a non significant mascot score) and from MCF10A cells EVs (Table 2b). These observation suggests that histone H1.2 might represent a potential marker for LN involvement and metastatic potential. Recent studies suggest histone H1.2 phosphorylation may be useful as a clinical biomarker of breast and other cancers because of its ability to recognize proliferative cell populations. Both MCF7 (expressing an allelic variant A142T) and MDA231, have a greater number of histone H1.2 phosphorylations when compared to MCF10A cell line²². Curiously, phosphorylation of histone H1.2 at S173 increases during the M phase relative to the S phase, suggesting that this event is cell cycle-dependent and may serve as a marker for proliferation of cancer cells during BC invasion^23,24. Also, histone H1.2 is a novel component of the nucleolar organizer regions during mitosis²⁵ and H1.2 depletion was observed in a human BC cell line caused cell cycle G1-phase arrest²⁶. Indeed, a higher mitotic index (≥ 7) in primary tumors is significantly associated with LN involvement²⁷ and higher mitotic indices accurately predict axillary LN involvement at operation²⁸.

Although we have identified two potential biomarkers possibly derived from EVs, our study does suffer from a number of recognised limitations. Firstly, ultracentrifugation is insufficient to purify EVs from other contaminants²⁹. For example, co-isolation of high-density lipoprotein and other particles with EVs isolated from blood by density gradient centrifugation has been reported^29,30 suggesting that the biomarkers we identified might not be associated with EVs but with a constituent of another particle type such as a lipoprotein. Secondly, as mentioned above, exosomes are defined as ∼30–100 nm vesicles that originate from MVB. In contrast, microvesicles or shedding vesicles are generally larger (>200 nm), more variable in shape and density and arise from the plasma membrane. The size of the particles we isolated ranged from 76.7-213.4 and 73.8-192.3 nm, for samples CF5 and CF37, respectively and for all the samples between 12.3-298.4nm (Figure 1 and original NTA files) possibly correspond to low-density lipoproteins which have the same size as EVs³¹. Moreover, it is unlikely that EVs would contain a histone (which are normally confined to DNA in the nucleus). However, Thakur et al., claim to have identified genomic DNA in EVs by electron microscopy (EM) though the EM image is not of sufficient magnification to allow for an accurate morphologic analysis and may simply represent cellular debris or apoptotic bodies or even unspecific staining^32,33. Additionally, it is also unlikely HCG1745306 isoform CRA-a, would be present in EVs and it may simply be a precipitant similar to the α-globin seen in β-thalassemia³⁴. Therefore our current data does not support the idea that these biomarkers derived from EVs and could in fact be blood-derived extracellular proteins.

Figure 1. Nanosight (NTA) analysis for samples C7, CF37, CF5, CF110, CF1, CF25, CF27 and CF33.

Nevertheless, a strength of our study is that samples were drawn from those with confirmed non-metastatic and metastatic disease at different sites and so are likely to be representative patients.

Conclusion

In conclusion, our observations suggest the possibility that HCG1745306 isoform CRA-a, and histone H1.2, irrespective of their origin, could represent potential biomarkers for the detection of early ER+ BC. Further work in a larger cohort of patients is clearly needed to confirm these initial findings.

Data availability

Dataset 1: The mass spectrometry analysis results from all patient samples 10.5256/f1000research.14129.d203204³⁵