Exosomal mRNA Cargo are biomarkers of tumor and immune cell populations in pediatric osteosarcoma

Abstract

Osteosarcoma is the commonest malignant bone tumor of children and adolescents and is characterized by a high risk of recurrence despite multimodal therapy, especially in metastatic disease. This suggests the presence of clinically undetected cancer cells that persist, leading to cancer recurrence. We sought to evaluate the utility of peripheral blood exosomes as a more sensitive yet minimally invasive blood test that could aid in evaluating treatment response and surveillance for potential disease recurrence. We extracted exosomes from the blood of pediatric osteosarcoma patients at diagnosis (n=7) and after neoadjuvant chemotherapy (n=5 subset), as well as from age-matched cancer-free controls (n=3). We also obtained matched tumor biopsy samples (n=7) from the cases. Exosome isolation was verified by CD9 immunoblot and characterized on electron microscopy. Profiles of 780 cancer-related transcripts were analysed in mRNA from exosomes of osteosarcoma patients at diagnosis and control patients, matched post-chemotherapy samples, and matched primary tumor samples. Peripheral blood exosomes of osteosarcoma patients at diagnosis were significantly smaller than those of controls and overexpressed extracellular matrix protein gene THBS1 and B cell markers MS4A1 and TCL1A. Immunohistochemical staining of corresponding tumor samples verified the expression of THBS1 on tumor cells and osteoid matrix, and its persistence in a treatment-refractory patient, as well as the B cell origin of the latter. These hold potential as liquid biopsy biomarkers of disease burden and host immune response in osteosarcoma. Our findings suggest that exosomes may provide novel and clinically-important insights into the pathophysiology of cancers such as osteosarcoma.

Keywords: Exosome; MS4A1; Osteosarcoma; TCL1A; THBS1.

Fig. 1

(A) CONSORT diagram illustrating study cohort and samples obtained. (B) Experimental schema showing exosome extraction and CTC isolation. Green circles denote fractions expected to be enriched for exosomes (S1: first supernatant, E: exosome fraction), while blue circles denote fractions not expected to be enriched for exosomes (P1: first pellet, S2: second supernatant); CTC: circulating tumor cell fraction obtained via the ClearCell® FX.

Fig. 2

(A) CD9 immunoblots, and (B) semi-quantitative comparison of the relative protein expression of CD9 in various fractions obtained from the exosome extraction protocol (P1, S1, S2, E), among non-cancer controls, and pre-chemotherapy and post-chemotherapy samples from osteosarcoma patients. Significantly higher CD9 protein expression was observed in exosome fractions of pre-chemotherapy samples compared to all prior fractions from the extraction process (* = P<0.05). (C) Representative transmission electron micrograph (TEM) of an exosome-enriched fraction isolated from the blood of an osteosarcoma patient (scale bar: 0.5 µm, case O3 (post-chemotherapy)). (D) Dot plot of exosome diameters measured on TEM images from non-cancer controls, pre-chemotherapy and post-chemotherapy osteosarcoma patients; one-way ANOVA; significant post-hoc tests between groups annotated (**** = P<0.0001).

Fig. 3

(A) Heatmap of normalized log2 counts of 780 tumor signaling-related genes among matched tumor, exosome and CTC samples from 4 osteosarcoma patients, demonstrating distinct clustering of tumor specimens from CTCs and exosomes. Rows represent genes and columns represent samples, unsupervised Pearson clustering. (B) Venn diagram of genes differentially expressed in tumors versus exosomes (left), and in tumors versus CTCs (right); names of relatively downregulated genes are colored blue, and upregulated genes colored red. One gene (CD14) was upregulated in exosomes but downregulated in CTCs, as compared to tumors. (C) Box plots of nCounter Tumor Signaling 360™ pathway signature scores for top 3 most upregulated pathways in exosomes and CTCs, and top 3 most upregulated pathways in tumors of osteosarcoma patients.

Fig. 4

Volcano plots of differentially expressed genes in exosomes from (A) osteosarcoma patients before neoadjuvant chemotherapy (O1-O7) versus non-cancer controls (C1-C3), and (B) osteosarcoma patients after neoadjuvant chemotherapy (O1-O5) against a baseline of matched exosomes before neoadjuvant chemotherapy from the same osteosarcoma patients (O1-O5). Individual genes are plotted by their -log10(p-value) and log2 fold change from baseline; significantly up or down-regulated genes are plotted at the top of the plot above the horizontal lines, and highly differentially expressed genes are plotted to either side. Horizontal lines indicate various adjusted p-value thresholds. Individual log2 counts of the three genes listed in Table 1 plotted for (C) control, pre- and post-chemotherapy samples, and (D) tumor, pre- and post-chemotherapy samples. Pooled normalization was applied, and annotated for disease stage at diagnosis and relapse status (C1-C3: control (Control E) group, O1-O5: pre-chemotherapy (E1) as well as post-chemotherapy (E2) groups). Means and standard errors of measurement are shown by the bars and whiskers. Data points for case O5 are marked, which had persistently high expression of THBS1 before and after neoadjuvant chemotherapy. (E) Representative THBS1 immunohistochemical stains of the primary tumor of case O5 corresponding to timepoint E2, as well as subsequent relapse tumors at bony and pulmonary sites (scale bar: 20 µm).

Fig. 5

Boxplots of immune cell type scores for neutrophils, macrophages and B cells, of (A) 7 pairs of osteosarcoma tumors and exosomes from peripheral blood at diagnosis (E1), and (B) peripheral blood exosomes of non-cancer controls (control E) and osteosarcoma patients at diagnosis (E1). (C) Representative immunohistochemical stains of CD20 and TCL1A in osteosarcoma initial biopsy specimen and relapse tumors of a case of metastatic relapsed disease (case O5) and in the initial biopsy and post-chemotherapy resected tumor specimens of a case of loco-regional disease (case O3) (scale bar: 20 µm).