CTC selection during cancer progression and treatment pressure
The analysis of the transcriptomic profiles of the nine CTC lines using Affymetrix HG-U133P microarray chips clearly separated them in four distinct groups: CTC-MCC-41, CTC-MCC-41.4, CTC-MCC-41.5 [ABFG], and CTC-MCC-41.5 [CDE] (Fig. 1a). We then compared the transcriptomic profiles of the pre-treatment CTC-MCC-41 line and of the other eight CTC lines (CTC-MCC-41.4 and CTC-MCC-41.5[A-G]) to understand the treatment impact on CTC clonal selection (Fig. 1b and Additional file 2: Tables S1–2). The significantly higher number of upregulated genes in the eight cell lines obtained after treatment initiation (Fig. 1c) suggests that they acquired properties to adapt and resist to treatment. For instance, genes involved in the mTOR and PI3K/AKT signaling cascades, which are implicated in cancer development by coordinating cell growth, survival and proliferation, and in resistance to chemotherapy, were upregulated in the post-treatment CTC lines (Fig. 1d). Colon cancer-specific mortality is higher in patients with tumors harboring mutated PIK3CA than wild type PIK3CA [8]. All our CTC lines harbored wild type PIK3CA and AKT [6], but these signaling pathways were deregulated. Some studies demonstrated that PI3K/mTOR pathway inhibitors could be used in primary and metastatic colorectal cancer [9]. We recently reported that CTC-MCC-41 cells also respond to mTOR and AKT inhibitors, suggesting these therapies are effective even in the absence of mutations [10].
Conversely, we did not observe many differences between the CTC-MCC-41.4 line, obtained after the last treatment, and the seven CTC-MCC-41.5 [A-G] lines obtained before the patient’s death (Additional file 2: Table S3). This suggests that, without drug pressure, the rapid cancer worsening was not link anymore to a clonal evolution but is due to natural disease progression with the replication of the CTC clones already selected under treatment. Indeed, the seven last CTC lines seem to be already present in the pooled CTC-MCC-41.4 line and they have been selected in the in vitro culture from the last blood sample. However, we could clearly segregate the last seven CTC cell lines (CTC-MCC-41.5 [A-G]), in two groups, [ABFG] and [CDE] (Fig. 1a), with different gene expression profiles (Fig. 1e). Most of the significantly deregulated pathways were involved in metabolism signaling (Fig. 1f), including xenobiotic metabolism. This suggests that detoxification mechanisms were induced upon exposure to anti-cancer drugs, as indicated by the deregulation of the irinotecan/SN38 pathway specifically in the [CDE] group. Furthermore, lipid metabolism upregulation appeared to be more represented in the “CDE signature”. Lipid metabolism is a key function on the basis of the enrichment of different signaling cascades leading to energy metabolism. Since Warburg’s work, metabolic reprogramming is one of the main hallmarks of cancer cells and plays a critical role in the continued tumor growth and progression and is driven by a complex interplay between the tumor mutational landscape, epigenetic modifications, and microenvironmental influences [11]. This topic is actively studied and a high-throughput metabolic-based assay was developed for rapid detection of rare metabolically active disseminated tumor cells in pleural effusion of lung cancer [12].
Cytidine deaminase as a drug resistance biomarker
Besides deregulation of the irinotecan/SN38 pathway in the [CDE] group, the gene encoding cytidine deaminase (CDA) was deregulated in the post-treatment CTC lines (Additional file 2: Table S2). This finding in a patient treated with 5-FU, a pyrimidine analogue, is interesting because CDA maintains the cellular pyrimidine pool by catalyzing the hydrolytic deamination of cytidine and deoxycytidine to uridine and deoxyuridine. Some studies showed that in patients treated with direct cytidine analogues, such as gemcitabine and cytosine arabinoside, CDA overexpression might be a marker of resistance [13]. Analysis of CDA expression in colorectal cancer using publicly available data [14] indicated that it was significantly downregulated in colon adenocarcinoma compared with normal colon (Fig. 2a). However, CDA was strongly upregulated in the CTC-MCC41.4 cell line obtained directly after failure of the first- and second-line 5-FU-based treatments (RT-qPCR analysis in Fig. 2b) and also, to a lower extent, in the CTC lines (CTC-MCC-41.5 A-G) obtained just before death, compared with the pre-treatment CTC-MCC-41 line.
As CDA is secreted in the extracellular compartment (https://www.uniprot.org/uniprot/P32320) and can be detected in blood, we quantified CDA concentration by ELISA in conditioned medium from the nine CTC-MCC lines. As observed for the CDA gene, CDA protein level was highest in CTC-MCC-41.4 cells (Fig. 2c). These findings suggest that in this patient, CDA was directly produced and secreted by resistant and aggressive CTCs in response to 5-FU-based chemotherapy. Therefore, CDA might represent a candidate plasmatic biomarker to monitor 5-FU efficacy and resistance development.
Aldolase B as a marker to identify CTCs released only by liver metastases
Comparison of the signatures of the two CTC-MCC-41.5 sub-groups ([ABFG] and [CDE]) (Additional file 2: Tables S4–5) highlighted ALDOB upregulation in the [ABFG] group (Fig. 2d). This gene encodes the aldolase B enzyme (fructose-bisphosphate aldolase B or liver-type aldolase), one of three isoenzymes (A, B, and C) of the class I fructose 1,6-bisphosphate aldolase enzyme (EC 4.1.2.1) that plays a key role in glycolysis and gluconeogenesis. This enzyme is preferentially expressed in the liver and at lower extend in the kidney and the small intestine. ALDOB downregulation correlates with poor overall survival in liver and gastric cancer [15, 16], whereas ALDOB overexpression in colorectal cancer has been associated with poor overall survival and epithelial-to-mesenchymal transition promotion [17]. Colorectal cancer is known to preferentially metastasize (∼70% patients) to the liver [18], which is the main organ for glycogenesis and gluconeogenesis. In addition, this specific patient had liver metastases. Comparison of ALDOB expression in different tissues using publicly available data showed that: (i) ALDOB was strongly expressed in normal liver compared with normal colon, (ii) ALDOB expression was comparable in primary colon adenocarcinoma and normal colon samples, and (iii) ALDOB was upregulated in colon cancer liver metastases compared with primary tumors (Fig. 2e). Bu et al. showed that during liver colonization, colon cancer cells undergo metabolic reprogramming by upregulating ALDOB [19]. This enhances fructose metabolism and promotes the growth of colon cancer liver metastases. These data suggest that the [ABFG] lines were derived from CTCs released by liver metastases. It is thought that CTCs are released by the primary tumor and/or metastases; however, to our knowledge, this is the first time that this could be demonstrated in a patient.
