Isolation of EV from metastatic cancer patients

Based on the established protocol for the isolation of total MV and Exo from peripheral blood samples, we aimed to investigate whether EV counts as well as the distribution of MV populations differs in cancer patients compared to tumor-free controls. EV were isolated from 21 cancer patients with metastatic disease including patients with primary lung cancer (n=8), breast cancer (n=4), colorectal cancer (n=4), ovarian cancer (n=1), melanoma (n=3) and glioblastoma (n=1). Tumor-free volunteers (n=14) were used as negative controls. The median patient age at the time of sample acquisition was 64 [IQR 59 - 74] years and the median age of the controls 52,5 [IQR 43,25 – 68,5] years with a p-value of 0,03517 (Wilcox two-sided rank test) indicating that patients were slightly older compared to the controls.

Fig. 42: Isolation and characterization of EV from peripheral blood of cancer patients A, Total EV were isolated from peripheral blood of tumor (n=21) and control (n=14) patients and EV yields determined by Lowry assay. Lines display the median of the samples. B, The isolated MV were further characterized for the established blood cell markers by flow cytometry (*p<0,05).

Nonetheless, comparison of EV yields between control and patient samples showed no difference in the amount of MV present in blood (patients: median 30,98 [IQR 18,12-47,43], controls: median 31,36 [IQR 14,98-59,63]) with a p-value of 0,6778 (Wilcox one-sided rank test). In contrast, Exo counts seemed to be slightly increased in tumor patients (median 25,98 [IQR 21,64-31,91]) compared to the controls (median 18,81 [IQR 9,57-31,85]), although with a p-value of 0,219 (Wilcox one-sided rank test) this did not reach statistical significance yet (Fig. 42A). Since none of the putative tumor markers was reliably expressed on T-Exo in in vitro screenings (Fig. 33), it was not possible to elucidate if this increase was due to an increased amount of T-Exo in blood. In case of MV, the established markers remained unchanged in both samples for platelet-derived (CD62P⁺, p=0,1632), erythrocyte-derived (CD235a⁺, p=0,4569) and myeloid cell-derived (CD11c⁺, p=0,7233) MV, whereas we

observed a slight increase in leukocyte-derived (CD45⁺) MV in tumor patients compared to the controls (p=0,01297, all Wilcox one-sided rank test) (Fig. 42B).

Next, we investigated if it is possible to detect T-MV in peripheral blood based on the previously established MV-associated tumor markers (Fig. 43). EpCAM-positive MV were only detected at very low levels (median 1,6% of total MV) in 15 out of 19 tumor patients and 5 out of 14 control patients. Although this represented a significant increase in patient samples (p=0,01262, Wilcox one-sided rank test), the low number of EpCAM-positive MV might explain the failure of EpCAM-based MACS for T-MV as described in 3.3.2. In contrast, neither MUC1 nor EGFR were expressed in any of the tested control samples (0/9), but positive in 5 out of 19 patients for MUC1 and 2 out of 18 patients for EGFR. However, this did not reach statistical significance.

Fig. 43: Tumor markers on MV derived from metastatic cancer patients

Whole MV were isolated from peripheral blood of metastatic cancer and control patients and analyzed by flow cytometry for the expression of the previously defined tumor markers EMMPRIN (A), EpCAM (B), EGFR (C) or MUC1 (D). Lines display the median±IQR. Significance was calculated with a one-sided Wilcox rank test.

Surprisingly, there was a high increase in the number of EMMPRIN-positive MV in tumor patients (median 31,6% of total MV) compared to tumor-free controls (median 11,2% of total MV) with a p-value of 8,633 ∙ 10^-5 (Wilcox one-sided rank test). To exclude that this finding was due to the higher age of the patients compared to the control group and further clarify the nature of the EMMPRIN-positive MV, we isolated MV from peripheral blood of five metastatic breast cancer patients for whom we had earlier defined possible tumor markers on breast cancer-derived T-MV in vitro. The median age of the patients at the time of sample acquisition was 51 [interquartile range (IQR): 49,3 – 57,0] years and the median age of 5 matched tumor-free controls 47 [IQR: 42,5 – 54,5] years with a p-value of 0,5196 (Wilcox two-sided rank test).

Fig. 44: Detection of T-MV in peripheral blood of metastatic breast cancer patients A-B, Total MV from five metastatic breast cancer patients and matched controls were analyzed by flow cytometry (A) to determine the percentage of EMMPRIN-positive MV (%positive events - % isotype control) (B). The line indicates the mean of the samples. C, Corresponding histograms for the EMMPRIN expression on total MV from breast cancer patients and controls with the specific signal in green and the corresponding isotype control in grey. D, Co-localization of the tumor markers EMMPRIN and MUC1 on MV from two metastatic breast cancer patients compared to matched controls as shown in density plots (flow cytometry).

The number of EMMPRIN-positive MV in peripheral blood was measured by flow cytometry and was found again to be significantly higher in patient-derived MV samples (median 14,7 [IQR 14,4-26,5] % of total MV) compared to controls (median 2,3 [IQR 2,3-6,2] % of total MV) with a p-value of 0,01366 (Wilcox one-sided rank test) as shown in Fig. 44A+B. Since our previous results had demonstrated that EMMPRIN can also be expressed on benign cells, e.g. platelets or myeloid cells (see Fig. 38 and Fig. 39), we investigated whether the EMMPRIN-positive MV additionally express other tumor markers which would argue for their origin from tumor cells. For this reason, we performed double stainings for EMMPRIN and MUC1 by flow cytometry. MUC1, also known as CA 15-3, is often found at high levels in the blood of hormone receptor-positive breast cancers (Park et al, 2012) and was already shown to be present on their released MV (Fig. 32). While two of the further investigated four breast cancer patients were negative for MUC1 on their MV and therefore could not be used for double stainings, the other two patients showed a co-expression of MUC1 with EMMPRIN on 34,2% or 18,7%, respectively, of all MV present in blood (B). Taken together, the results suggest that T-MV can indeed be found in high numbers in the blood of metastatic cancer patients and can be identified by co-localization of the established markers, including EMMPRIN and MUC1.