Detection of circulating sarcoma tumor cells using a microfluidic chip-type cell sorter

Analyses of circulating tumor cells have been shown to be effective for the detection of cancer relapse and prognosis prediction. However, research regarding its utility in sarcoma remains scarce. In this study, the microfluidic chip-type cell sorter On-chip Sort was used to construct a system for detecting circulating sarcoma cells (CSCs). A pilot study using normal fibroblast or sarcoma cell lines was designed to establish a reliable protocol to separate CSCs by On-chip Sort. A single CSC was separated and recovered from 10 ml of whole blood from a patient with locally advanced myxofibrosarcoma. The nonsynonymous mutation for KMT2B p.Ile2602Val identified in the formalin-fixed paraffin-embedded tumor sample was also confirmed in the CSC. Use of the developed protocol may allow CSCs to become an early predictor for metastasis and recurrence of sarcoma. Further, it may aid in optimizing post-operative therapies for patients without metastasis.


Results
Protocol for CSC enrichment, enumeration, sorting, and sequencing. An overview of the CSC analyses used in this study is shown Fig. 1. Blood cell depletion was carried out by autoMACS Pro Separator using CD45 MicroBeads and CD235a MicroBeads. The CD45 antigen is expressed in all cells of hematopoietic origin except erythrocytes, platelets, and their precursor cells. The CD235a antigen (glycophorin A), a single-pass transmembrane glycoprotein, is expressed in mature erythrocytes and erythroid precursor cells.
The remaining cells were fixed and stained with antibodies specific for sarcoma, vimentin, or white blood cells (WBCs), such as CD45 and CD14. The CD14 antigen, a component of the LPS receptor complex, is strongly expressed in most monocytes and macrophages and weakly expressed in neutrophils and some myeloid dendritic cells. Conversely, vimentin is expressed in a wide range of cell types. These include stromal cells, fibroblasts, endothelial cells, and neuronal precursor cells. It was hypothesized that CD45/14 (-)/vimentin (+) fractions in peripheral blood may specifically contain CSCs.
CSC enumeration and sorting were carried out using the On-chip Sort system (On-chip Biotechnologies). Genome sequencing was performed after whole-genome amplification (WGA) of CSCs (Fig. 1).
A pilot study using the human fibroblast cell line BJ was designed as follows. Five hundred BJ cells were spiked in 3.0 × 10 5 of WBCs. The sample was then fixed and stained with anti-CD45 mouse monoclonal antibody (mAb) conjugated to allophycocyanin (APC), anti-CD14 mouse mAb conjugated to APC, 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI), and anti-vimentin Alexa Flour 488-conjugated rabbit mAb. We intended to enrich spiked BJ cells (skin fibroblasts) by reducing WBCs (CD45-positive), macrophages, and monocytes (CD14-positive). To completely remove macrophages and monocytes, we utilized an APC-conjugated anti-CD45 Ab combined with an anti-CD14 Ab. A CD45/14 (−)/vimentin (+) fraction from the sample was purified with the On-chip Sort system. Sorting was repeated three times to enrich CSCs. After purification, a total of 326 cells were gated as CD45 (−)/vimentin (+), and aliquoted into 10 tubes (containing ~30 cells per tube) ( Fig. 2A). One of those tubes was subjected to WGA.
The short tandem repeats (STRs) determined in the WGA sample were used to investigate the composition of the sorted cells. The STR locus D5S818 of the BJ genome contains 12 repetitions of "AGAT" in both alleles, whereas WBCs have 10 repetitions in one allele and 13 repetitions in the other. As shown in Fig. 2B, Sanger sequencing of the polymerase chain reaction (PCR) products for the D5S818 locus revealed that 85.7% of the sorted fraction were identified as BJ cells.
WBC removal using autoMACS Pro Separator. The necessity of WBC depletion from the whole blood sample before running On-chip Sort was next explored. Approximately 50 cells of the rhabdomyosarcoma cell line RH30 were spiked into 2 ml of whole blood collected from a healthy volunteer. One sample underwent red blood cell (RBC) lysis without a negative selection for WBCs. The sample was then fixed, stained, and run through On-chip Sort. However, RH30 cells were not well-separated from the WBC group (Fig. 3A). In contrast, RH30 cells were clearly identified after removing WBCs from the sample using autoMACS Pro Separator (Fig. 3B). Protocol for CSC enrichment, enumeration, sorting, and sequencing. An overview of CSC analysis in this study. The whole blood obtained from a sarcoma patient was enriched using autoMACS Pro Separator (Step 1). CSCs were fixed and stained with antibodies specific for sarcoma or WBCs (Step 2). CSC enumeration (Step 3) and sorting (Step 4) were carried out using the On-chip Sort system. Genome sequencing was performed after WGA of CSCs. The permission for the usage of image of On-chip Sort was obtained from On-chip Biotechnologies.
Forty-seven cells gated as CD45 (−)/vimentin (+) were collected and divided into three tubes. One of those tubes was subjected to WGA. Sanger sequencing was performed to confirm the TP53 c.817C > T DNA mutation (protein mutation: p.Arg273 Cys) in the amplified DNA, which is known to be present in the RH30 genome. The electrophoretogram of the sorted cells was the same as that of bulk RH30 cells (Fig. 3C). Thus, depleting WBCs before loading the sample onto On-chip Sort improved the separation accuracy for sorting spiked sarcoma cells from whole blood samples.
Method for gating CSCs using WBCs from the same patient on On-chip Sort. To construct an appropriate gate for vimentin, RH30 was always analyzed as a positive control cell line in parallel to the specimens of interest. The CSC gates for the flow cytometer were set to contain ≥90% of RH30 cells (G1) and no WBCs of the patient (G2). WBCs were separated in advance from 10 ml of whole blood using autoMACS Pro Separator. The first round of sorting was performed using the G1 gate, and the second and the third rounds were performed using the G2 gate (Fig. 4).

Detection of CSCs in a myxofibrosarcoma patient.
A 60-year-old man with myxofibrosarcoma (histological grade: Fédération Nationale des Centers de Lutte Contre le Cancer grade 3, disease stage: American Joint Committee on Cancer/Union for International Cancer Control tumor-node-metastasis stage III) underwent preoperative chemotherapy, followed by surgical excision and post-operative chemotherapy. A blood sample was collected from the patient before the preoperative chemotherapy, 3 months post-, and 9 months post-surgery. No clear metastases were observed at any point during blood sampling (Fig. S1).
Erythrocytes and WBCs were removed from the collected blood specimens. The samples were then fixed and stained with antibodies for CD45, CD14, DAPI, and vimentin. CSC enumeration and sorting was performed using On-chip Sort. A single CSC was sorted at 3 months after surgery (Fig. 4), while no CSCs were detected before treatment or at 9 months after surgery (Fig. S2).
Formalin-fixed paraffin-embedded (FFPE) tumor tissue, normal tissue, CSC, and cfDNA sequencing. To determine whether alterations in the genomes of primary tumor cells can be detected in CSCs and cfDNA, we analyzed genomic alterations in single CSCs and cfDNAs three months after surgery and compared them with those found in the primary tumor FFPE sample. Genomic DNA of tumor or normal tissues isolated from the FFPE and peripheral blood specimen of the patient were subjected to extensive sequencing for cancer-related genes using the TOP panel 23 . A subsequent bioinformatics analysis revealed cancer-associated somatic mutations, including KMT2B c.7804A > G DNA (protein: p.Ile2602Val) and MGA c.3628C > G DNA (protein: p.Arg1210Gly) with allele frequencies of 13.06% and 6.12%, respectively (Table 1).  (Table 1). To verify that the KMT2B mutations observed in cfDNA at low VAFs were true positive mutations, targeted deep sequencing was performed in two giant cell tumor of bone (GCTB) specimens that did not have KMT2B c.7804A > G in the original tumors. The KMT2B c.7804A > G mutation was also found in the GCTB tumors at VAFs of 0.19% and 0.20% (Table 1). Therefore, the KMT2B mutations found at low VAFs were likely to be sequence errors.

Discussion
The current study established a new protocol for CSC detection using a microfluidic chip-type cell sorter, the On-chip Sort. Several reports have described detection of CSCs using different devices, such as a size-exclusion microfiltration system 24 and flow cytometry 25,26 with specific markers. However, the utility of size-exclusion is limited by the lack of a histology-specific marker. Flow cytometry is limited due to the heterogeneous expression of target markers, which may affect the detection sensitivity. Even specific markers, such as CD99 for Ewing's sarcoma, show background expression on normal hematopoietic cells 27 .
As there were no standard markers suitable for CSC isolation, vimentin was used to target mesenchymal cells 28 . However, WBCs are also known to express vimentin to some extent. Thus, the number of WBCs was first reduced using a negative selection with the autoMACS Pro Separator system. Then, the CD45 (−)/vimentin (+) fractions were separated using On-chip Sort. Simultaneous analyses of RH30 as a vimentin (+) control and WBCs as a negative control from the same patient allowed for precise gating of the target fraction. Theoretically, the current protocol may be applicable for the isolation of any type of CSCs.
A previous report used real-time PCR to confirm the fusion genes of CSCs 29 . Although fusion genes are found in about one-third of sarcomas, no specific genetic abnormalities can be identified in the remaining two-thirds of sarcomas, including myxofibrosarcoma. Somatic mutations identified by the TOP panel can be utilized to confirm any type of CSCs. Reports on sarcoma cfDNAs have been published [30][31][32] , and efforts have been made to identify sarcoma mutations in cfDNA by improving sequencing techniques 33 . However, cfDNA remains difficult to detect in pre-metastatic cases. The current study showed that, in contrast to the CSC analysis, cfDNA was unable to detect the somatic KMT2B mutation found in the sarcoma. MicroRNAs have also attracted interest in the study of STS 34,35 , although their utility in clinical practice has not been demonstrated. By improving the sorting method, CSCs can be identified in patients before metastasis. These findings suggested that CSCs www.nature.com/scientificreports www.nature.com/scientificreports/ might be an early predictor of metastasis and recurrence in sarcoma and could help inform decisions concerning post-operative radiation therapy and chemotherapy for patients without metastasis. Further studies are needed to compare the limit of detection between CSCs and cfDNA analyses.
Presence of CTCs (three cells or more) was shown to predict shorter progression-free survival/OS for colon cancer patients 36 . While one CSC was detected at 3 months after surgery, the patient in the current study did not demonstrate any signs of metastases after 9 months of surgery. This may suggest that a few remaining CSCs are insufficient to colonize any metastatic sites. Quantitative evaluation of CSCs with the current protocol might help to stratify the prognosis of patients with few CSCs. The accuracy of separating CSCs can be further improved by adding other selection windows, such as CD99 for Ewing sarcoma or MDM2 for liposarcoma. The clinical utility of the current improved protocols must be tested in larger cohorts.
One of the limitations of this study is the sample preparation for the pilot study. The 65% recovery in this pilot experiment using the BJ cell line was relatively lower than that of the other platforms because the number of manually prepared BJ cells used for the spike was not 500. The other pilot experiment using the RH30 cell line shows . CSC sorting in a 60-year-old man with myxofibrosarcoma. A CSC-specific gate including ≥90% of RH30 cells (G1) was created using simultaneously prepared RH30 cells as a vimentin (+) control. A gate that included no WBCs from the patient (G2) was created using the patient's WBCs as a vimentin (−) control. WBCs were separated in advance from 10 ml of whole blood by autoMACS Pro Separator. The first round of sorting using the G1 gate resulted in 51 cells. The second and the third rounds using the G2 gate resulted in four cells and one cell, respectively. Patients and tumor samples. This project was approved by the ethics review board of Juntendo University Hospital (No. 2018169). Written informed consent was obtained from the patients whose samples were analyzed in the present study. Samples were collected from a myxofibrosarcoma patient who received chemotherapy and two GCTB patients at the Department of Orthopedic Surgery in Juntendo University Hospital. A 10 ml sample of whole blood and FFPE were collected at the time of diagnosis. In the myxofibrosarcoma patient, blood samples were collected at three points: before treatment was started, 3 months post-, and 9 months post-surgery. In addition, whole blood was collected from healthy participants. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Immunomagnetic enrichment. Blood samples (10 ml) were collected with blood collection tubes (EDTA-2K) and used within 3 h. Whole blood was separated into two 15 mL Conical Centrifuge Tubes (Thermo Fisher Scientific, Waltham, MA, USA) and 5 ml of autoMACS Pro Separator Running Buffer was added to each sample. The samples were then centrifuged at 400 g for 10 min to separate the plasma without damaging the buffy coat. CD45 Micro Beads were added to each 5 ml sample at a concentration ratio of 1:20. The sample was then stirred by MACSmix Tube Rotator (Miltenyi Biotec, Bergisch Gladbach, Germany) for 15 min. After stirring, Running Buffer was added to bring the sample up to 13 ml to flush the CD45 MicroBeads attached to the tube wall. The samples were then centrifuged at 400 g for 5 min to separate the plasma without damaging the buffy coat. The WBCs were then separated out using autoMACS Pro Separator. The negative fraction was mixed with 4 ml RBC Lysis Buffer (TONBO Bioscience, San Diego, CA, USA) and 28 ml UltraPure DNase/Rnase-Free Distilled Water (Thermo Fisher Scientific). The samples were then stirred using MACSmix Tube Rotator for 10 min. The samples were then centrifuged at 400 g for 5 min to separate the supernatant. Running buffer (10 ml) was added to the samples, and the samples were centrifuged again at 400 g for 5 min and the supernatant was removed. Both samples were then mixed together and running buffer was added to bring the sample up to 500 µl. PE anti-human CD235a (Glycophorin A) antibody was also added at a concentration of 1:50. The sample was incubated for 10 min. Anti-PE MicroBeads (Miltenyi Biotec) were added to the sample at a concentration of 1:5 and incubated for 10 min. Running buffer was added up to 5 ml and the sample was then placed in autoMACS Pro Separator. The negative fraction was centrifuged at 300 g for 10 min and the supernatant was removed. Running buffer was added to bring the sample up to 500 µl, and the sample was centrifuged at 300 g for 10 min and the supernatant was removed. Running buffer was added up to 100 µl to load the sample onto On-chip Sort. WGA and PCR for genotyping. Cells sorted by On-chip Sort were transferred from the collection reservoir of the chip to a 200 μL PCR tube and centrifuged at 400 g for 10 min. Excess liquid was removed after centrifugation, leaving 2 μL of the liquid. WGA was performed using the SMARTer PicoPLEX WGA Kit (Takara Bio Inc., Shiga, Japan) following the manufacturer's protocol. The amplified products were subjected to Sanger sequencing or next-generation sequencing (NGS). www.nature.com/scientificreports www.nature.com/scientificreports/ Mutational profiling of primary tumor by TOP. The detailed protocol and the content of gene panel were described in previous paper 23 . In brief, gDNA (500 ng) of tumor FFPE and normal blood samples were subjected to target fragment enrichment using a SureSelectXT Custom kit (Agilent Technologies). Custom-made probes were designed to hybridize and capture the gDNA of the target genes listed by TOP. Massive parallel sequencing of the isolated fragments was performed using a HiSeq. 2500 (Illumina, San Diego, CA, USA).