MIF/CD74 axis is a target for novel therapies in colon carcinomatosis

In-phase contrast microscopy showed both tubular-branched and more round shaped 3D structures present in C1, while C2 was mainly composed of round-shaped cellular aggregates (Additional file 2: Figure S1).

After paraffin inclusion, C1 organoids exhibited glandular structures lined by pseudostratified columnar cells with central fibrinoid core and dirty necrosis along with roundish solid aggregates of not aligned tumour cells. These features mirrored those present in the corresponding histologic sample (Fig. 1a). In C2 organoids the cells exhibited adhesion loss and were organized in ring and/or ribbon-like structures. The corresponding sample showed solid nests of poorly differentiated cells with eosinophilic cytoplasm surrounded by desmoplastic stroma infiltrating omental fat (Fig. 1b). Both organoids expressed Cytokeratins (CK) A1-A3, −19, −20 (Fig. 1) and Claudin 4, and were negative for CK7 and Paired Box 8 (PAX8) (data not shown). C1 retained a high expression of Caudal Type Homeobox 2 (CDX2) compared to C2. Finally, according to the observed rapid growth, both organoids had a very high Ki-67 staining (Mib1) (Fig. 1).

By ISH, we investigated the expression of LRG5, the marker of the cell progenitor population located at the crypt-base [4, 5]. LRG5 RNA expression was observed in both organoids (Fig. 2). C1 displayed a gradient of LRG5 staining that was stronger in the peripheral sheet of epithelial cells compared to more luminal sheets. Conversely, C2 LGR5 was very focally expressed by some apical and peripheral organoid cells, resulting in a more heterogeneus staining. Transcription of LGR5 was also confirmed in their corresponding clinical FFPE materials (Fig. 2).

Taking advantage from the serum-free growth conditions of CRC-pc organoids, we profiled the secretome compartment to search for novel molecular candidates for therapy involved in CRC-pc development and coupled it with a classical pharmacological approach aimed at exploring the possible implications of these candidates in CRC-pc. Indeed, we also used C1 organoids for validating the results obtained from the use of C2.

We stated that these proteins playing a role in crucial biological processes were secreted by organoids and that they were present also in clinical samples. These studies support the concept that CRC-pc organoids may constitute a pre-clinical model for investigating the biology of CRC-derived peritoneal lesions.

The secretome composition of C2 organoids was investigated using a classical bottom-up proteomic analysis with LC-Orbitrap mass spectrometry. Of the 229 items, 191 (83%) were recognized as proteins with GO extracellular space by STRING (Fig. 3a). PANTHER assessed overrepresentation of proteins involved in Parkinson disease, glycolysis and pentose phosphate pathways (Fig. 3b). These results are consistent with the previous observation that functions related to glucose metabolism and mitochondria homeostasis are downregulated in neurodegeneration, including Parkinson’s disease (PD), but upregulated in colorectal cancer [21].

Analysis of the secreted proteins with annotations in the IPA database identified networks that were enriched in proteins involved in metabolism homeostasis, protein ubiquitination, cell cycle and DNA repair, and were significantly over-represented in cancer (Additional file 2: Figure S2).

Secretome composition of C2 organoids included proteins potentially able to mediate high metabolic demand, adaptive homeostasis along with enhancing stress resistance. One of them was the multifunctional cytokine MIF (Fig. 3c), the ligand of CD74 receptor. MIF/CD74 axis can promote cell survival by modulating glucose uptake and metabolism through the activation of AKT and AMPK proteins and is considered a potential target for therapy [22] (Fig. 3c).

MIF and CD74 were expressed in C2 organoids and in the corresponding tumour tissue (Fig. 4a and b). We then characterized key molecules involved in the downstream-signalling cascade of the MIF/CD74 complex. As expected C2 organoids exhibited AKT- and AMPK-specific phosphorylation (respectively at positions S473 and T172, Fig. 4a) and suppression of JNK activity due to MIF mediated inhibition of JAB1 (data not shown).

Since MIF/CD74 axis through AMPK activation accelerates glucose uptake, which enhances mitochondrial functions [23], we assessed whether the MIF/CD74 axis supports and maintains high levels of metabolism of C2 organoids. We have recently observed an extensive alteration of some crucial oncogenic signals and a decrease in Krebs cycle activity through the specific inhibition of MIF activity using 4-IPP in papillary and anaplastic thyroids carcinoma models [24]. In the work we demonstrated by MALDI-TOF analysis that 4-IPP can effectively recognize MIF as its target.

We expect that targeting MIF/CD74 axis in CRC-pc models could give similar results inducing a strong impairment in many crucial metabolic pathways and leading to a dramatic change of the homeostasis of colonic carcinomatosis. Conversely, we examined the effects of AMPK stimulation by using metformin, an AMPK activator that disrupts mitochondrial functions through inactivating mitochondrial respiratory chain complex I [25].

We evaluated the 4-IPP effects by targeting C2 organoids with various concentrations of inhibitor (10, 25, 50, 100 μM). Twenty-four hours after treatment with 4-IPP, C2 organoids appeared disaggregated (Fig. 4c left) and died within 48 h. The disaggregation of organoids was also observed after treatment with metformin that, on the other hand, induced only a slight increase of cell death after 120 h (Trypan blue assay, Fig. 4d).

Treatment with 50 and 100 μM 4-IPP activated ERK phosphorylation. In contrast, AKT and AMPK phosphorylation was abrogated (Fig. 4e). The decreased phosphorylation of the AKT-specific phosphorylation site on FOXO3 further demonstrated the suppression of AKT activity and possibly supported the impairment of C2 viability. Shorter 4-IPP treatments (3, 6 and 12 h) exhibited similar results (Additional file 2: Figure S3). Treatment of C2 organoids with metformin inhibited phosphorylation of mTOR, ribosome protein S6 and 4E-BP1 but did not inhibit ERK and AKT (Fig. 4f). Treatments with 4-IPP increased the activity of the stress-activated protein kinase, JNK, in a dose-dependent manner, explainable by the activation of a complete stress-related response (Fig. 4e). In parallel, a dramatic change in the anti-phosphotyrosine/phosphothreonine profile was observed probably due to inhibited phosphatase activities (Fig. 4g, left panel). Indeed, PP2A phosphorylation was switched off after 4-IPP treatment (Fig. 4h, left panel). Conversely, metformin treatment did not significantly modify phosphotyrosine/phosphothreonine profiles and phosphorylation of PP2A and PPP1A (Fig. 4g, and h, right panels).

These results suggested that inhibition of AKT and AMPK-related pathways has more pronounced effects in terms of cell survival and stress-related response rather than the implementation of AMPK/mTOR/S6/4EBP1 axis (Fig. 4i).

To investigate the metabolic effects of the two drugs, we analysed the cellular amount of some metabolites involved in glycolysis, tricarboxylic acid (TCA) cycle, fatty acids (FAs) and cholesterol synthesis in lysates from C2 organoids after 4-IPP and metformin treatments by isotope-dilution gas chromatography-mass spectrometry (GC-MS) analysis [15]. After 4-IPP treatment, glycerol-3-phosphate and pyruvate were elevated and citrate, fumarate, malate and succinate were reduced (Fig. 5 and Additional file 1: Table S3). Conversely, after metformin treatment, lactic acid and glycerol-3-phosphate were elevated and pyruvate, succinate, fumarate, malate and citrate were reduced. 4-IPP treated C2 organoids showed a moderate reduction in fatty acids and cholesterol synthesis, while exposure to metformin resulted in reduced fatty acid levels and cholesterol synthesis that were more pronounced than that observed in cells treated with 4-IPP (Fig. 5 and Additional file 1: Table S3). Metformin treatment reduced cholesterol synthesis (measured as the cellular amount of cholesterol precursor lathosterol, lanosterol and desmosterol), as well as the amount of cholesterol and membrane fatty acids which are part of phospholipids, structural molecule of the lipid bilayer. It is likely that the alterations to the cholesterol and fatty acid metabolism may be both the consequence as well as a mechanism to induce the mitochondrial dysfunction in tumour cells exposed to metformin. Moreover the increase of pyruvate followed by the strong impairment in the production of the principal metabolites involved in the TCA cycle in C2 organoids after 4-IPP treatment seems to be related to a mitochondria disruption provoked by the knock down of MIF activity.

Because mitochondria are known to produce significant amounts of ROS that may generate oxidative stress contributing to mitochondrial damage, affecting signal transduction pathways, cellular functions and viability, we studied the effects of the two compounds on ROS production (Fig. 6a). ROS-H₂O₂-Glo assay showed an increase of ROS production in C2 organoids after metformin and 4-IPP treatments (2–2.5 times). This result suggests that C2 organoids undergo genotoxic stress due to rising levels of ROS.

A mitochondrial-targeted probe which accumulates in mitochondria, depending on their membrane potential, was used to morphologically explore the mitochondrial network in C2 organoids under 4-IPP and metformin inhibition. Briefly, we monitored the mitochondrial membrane potential using JC1 probe, a cationic carbocyanine fluorescent dye that exhibits a potential-dependent accumulation in mitochondria: a fluorescent emission shift from green (~525 nm) to red (~590 nm) indicates increase of the membrane potential, while a decrease in the red/green fluorescence intensity ratio highlights mitochondrial depolarization. Our analyses showed that mitochondria were distributed broadly in the cytoplasm and characterized by a perinuclear punctuated network in untreated cells (Fig. 6b). Cells treated with 4-IPP showed mitochondria with an altered morphology indicated by changes in immunofluorescence staining patterns. Mitochondria appeared much less tubular and a diffuse cytosolic pattern of fluorescence was observed. Of note, treated cells also showed signs of nuclear destruction (Fig. 6b, left). Metformin treatment decreased the fluorescence of the probe and several cells displayed a negative fluorescence signal (Fig. 6b, right), indicating a clear loss of mitochondrial membrane potential, which is an important indicator of functional mitochondria. The results highlighted a mitochondrial depolarization after metformin treatment (Fig. 6c).

Using electron microscopy to examine the morphology of mitochondria in C2 organoids treated with 4-IPP, we observed signs of necrosis, including strong condensation of chromatin along a nuclear envelope. There, mitochondria were smaller than in non-treated cells with significant impairment of their structure. The number of lipid droplets in cytosol was smaller than in cells treated with metformin (Fig. 6D1-D3). In organoids treated with metformin, we found many large mitochondria with irregular contours and a modest number of cristae. These mitochondria contained multi-lamellar structures inside, and were aligned with residual lipid droplets accumulated inside the cytosol (Fig. 6D4-D6).

These results suggested that ROS production likely due to the loss/reduction of mitochondrial activity affected viability of C2 organoids (Fig. 6e). Specifically, 4-IPP treated C2 organoids showed autophagy structures (Fig. 6d) that are associated with necrosis and cell death.

To further evaluate the relevance of the results obtained through 4-IPP and metformin, we assessed whether 4-IPP and metformin could inhibit the MIF/CD74 signalling axis and AMPK, respectively, by determining their effects on the expression of targets also in C1 organoids. Treatments impaired the principal morphological features of C1 organoids, leading to loss of the spheroids organization (Fig. 7a and d). Western blot analysis confirmed that 4-IPP at 50 and 100 μM increased ERK-1/2 phosphorylation and down-regulated AKT and AMPK activity (Fig. 7b). Moreover in C1 organoids, 5 mM metformin inhibited the mTOR pathway, and downregulated AKT and ERK activity (Fig. 7e). As shown in Fig. 7c, the anti-phosphotyrosine and anti-phosphothreonine profiles of the 4-IPP-treated cells presented a significant increase in phosphorylated proteins profiles compared with control groups. In parallel, we observed the change of PP2A phosphorylation status indicating failed phosphatase activity (data not shown). Trypan blue assay also displayed that 4-IPP treatments at 50 and 100 μM, strongly impaired C1 organoids viability after twenty-four and forty-eight hours. On the other hand, after one-hundred and twenty hours, 5 mM metformin slightly affected C1 viability (Fig. 7d). Taken together, these results confirm the validity and reproducibility of treatments with 4-IPP and metformin in colon-cancer carcinomatosis-derived organoids.

Immunohistochemical expression of CD74 and MIF was studied in three additional peritoneal CRC-pcs (C3, C4 and C5), along with their corresponding normal colon samples (Fig. 8). In normal colon samples, CD74 was widely and strongly expressed by both normal epithelial cells and the surrounding reactive cell microenvironment. The lymphocytes were strongly CD74 positive. The expression of CD74 in neoplastic tumour cells was heterogeneous, ranging from strong cytoplasmic and membranous (samples C4 and C5) to weak granular cytoplasmic (sample C3). MIF was poorly expressed by normal epithelial cells. Conversely, all tumour cells exhibited cytoplasmic and membranous staining which ranged from weak to moderate and strong.