Organoids Reproduce Metabolic Alterations Of Colorectal Cancer: A Good Tool To Choose The Best Drug Based On Tumor Stage

Colorectal cancer is considered to be one of the most commonly diagnosed cancers and it also has one of the highest mortality rates among malignant tumors.

The Molecular Oncology group at the IMDEA Food Institute (Madrid) is especially interested in the implication of lipid metabolism in tumor progression. Among the hallmarks that describe cancer evolution, we can find energetic metabolism. Adjustments to energy metabolism are triggered to fuel the uncontrolled growth of cancer cells. Concretely, lipid metabolism represents an important source of energy as well as structural and biosynthetic resources that are fundamental for the carcinogenic processes.

We are especially interested in a family of enzymes, Acyl-CoA synthetases (ACSL), which catalyze the conversion of long-chain fatty acids to acyl-CoA. More explicitly, isoforms 1 and 4, together with the stearoyl-CoA desaturase-1 (SCD), are the rate-limiting enzymes that convert saturated into monounsaturated fatty acids. Both have been extensively reported to be involved in tumor progression. We previously reported that ACSL1, ACSL4, and SCD overexpression (ACSLs/SCD network) make up a lipid network that fuels migratory and invasive properties through an epithelial-mesenchymal transition (EMT) induction and is associated with an increased risk of relapse in colorectal cancer (CRC) patients (1,2).

Furthermore, we performed a deep study on the regulation of this network by miRNAs, small non-coding RNA molecules; and we provide novel evidence that miR-19b-1, miR-544a, and miR-142 are able to target the pro-tumorigenic lipid network ACSLs/SCD. Importantly, miR-19b-1 emerges as a potential noninvasive biomarker given its strong association with a better prognosis in stage II and III CRC patients, and its ability to inhibit the invasion of CRC cells is showing to be a promising therapeutic miRNA in CRC.

After studying our axis in two-dimensional cancer cell line cultures, we felt the need for more innovative models resembling the features of the original tumors. In this way, we became very interested in a new model system of cancer, a three-dimensional culture system called organoids.

This model system closely recapitulates the in vivo situation of the tissue of origin. In contrast to cell lines grown in two-dimensional cultures, three-dimensional organoid cultures display features of the original tissue in terms of architecture, the cell types present, and their self-renewal properties. This methodology was first established by Sato et al (3) in a long-term primary culture from mouse small intestinal crypts to generate epithelial organoids with crypt- and villus-like epithelial domains representing both progenitor and differentiated cells.

The epithelium of the adult small intestine forms a contiguous two-dimensional sheet: crypts and villi. New cells are added in the crypts and removed by apoptosis upon reaching the villi tips a few days later. Stem cells and Paneth cells at the crypt bottom escape this flow.
The organoids’ technology takes advantage of the intestinal epithelium self-renewing capacity. Organoids start from the Lgr5+ gut or duct epithelial stem cells forming symmetric cyst structures, which will eventually form budding structures resembling intestinal crypts. This process takes between 7 and 10 days; later splitting of organoids leads to single-cell dissociation and the formation of new organoids that could be indefinitely propagated through such passaging

Organoids can be easily manipulated by genetic engineering tools. Furthermore, one of the outstanding applications is high-throughput drug screening, which is not possible in in vivo systems such us mice or humans, which represents excellent physiological tools for the development of new CRC personalized treatments. In this way, we decided to get a more in vivo approach for the analysis of the lipid metabolism-related axis, ACSLs/SCD, by employing genetically-engineered intestinal mouse models. These organoids have the relevant mutations acquired throughout the different CRC stages (APC^fl/fl, KRAS^G12D/WT, P53^R172H/WT, and Smad4^fl/fl; corresponding to the tumor stages I through IV) named CRC-like organoids.

To check the status of the axis in CRC-like organoids, we measured the mRNA levels of the ACSL/SCD network members. ACSL4 mRNA levels clearly augmented throughout the organoids’ stages, correlating with the increased aggressiveness of the organoid. Conversely, ACSL1 levels were stable over the series, and SCD levels increased from the third stage henceforth.

In the case of miRNAs, miR-19b-1-3p expression was decreased in a stage-dependent manner, maintaining its good role during prognosis. The rest of the candidate miRNAs were also measured, though no statistically significant differences were found in its expression. Therefore, miR-19b-1-3p preserved its protective role, reflecting the ACSLs/SCD axis and its regulation involvement on CRC progression.

Since metformin, an AMPK activator used as antidiabetic treatment that has been associated to increased survival of cancer patients, was able to rescue the epithelial phenotype from the mesenchymal process caused by the overexpression of ACSL/SCD in CRC cells (1); we wondered what this drug’s effect would be during the different stages of tumor progression. Using MTT assays, a colorimetric assay to assess viability, we compared metformin action with the current chemotherapeutic drug 5-fluorouracil (5-FU) in a 48h treatment. Metformin was able to decrease CRC-like organoids’ viability in all stages at the same rate as 5-FU, without affecting wild-type (WT) organoids’ viability.

To further check the treatment’s scope, we analyzed not only the effect but also the potential reversibility of the treatment. We assessed the organoids’ recovery capacity after a 48h treatment plus the subsequent recovery of 72 additional hours in their growing media. Organoid metformin-treatment recovery was significantly lower compared to 5-FU in the first-stage organoids, but a greater recovery was observed in WT organoids; suggesting metformin could have a potential use as a chemotherapy drug during the first tumor phases. In all other stages, 5-FU showed a major effect, with lower recovery rates. Furthermore, we proved that metformin was able to downregulate the crypt stem cell biomarker LGR5 and the Wnt target genes’ expression in all CRC-like organoid stages, reaffirming its potential use in intestinal cancers. Metformin action was also stronger on ACSL4 and SCD-overexpressing first stages organoids, diminishing these axis components’ mRNA levels. This is in accordance with metformin’s greater action during this first stage.

Finally, we observed that metformin action in CRC organoids was not related to a Warburg-effect impairment since L-lactate levels were even higher in metformin-treated CRC-like organoids despite its antitumor progression effect. This increase was significantly higher in stage I organoids, coincident with the higher sensitivity to the drug in this stage of CRC-like organoids.