T-Type Calcium Channel Blocker Promises a Potent Role as an Anti-Cancer Drug in the 3D Cancer Spheroids

Increasing death incidences due to cancer and the resistance towards the treatment call for better alternatives or enhancement in the current mode of treatments. Studies have shown the possible role of T-type calcium channel (TTCC) in the enhanced proliferation of cancer cells. In vivo and in vitro studies have shown that blocking this channel reduces the tumour size and viability, respectively. Most of the studies are carried out either in monolayer cell culture or in an animal model which does not represent actual human physiology and hence the drug testing fails in clinical trials. Drug testing in cancer spheroids is a new emerging mode of study where the cancer spheroid with a hypoxic and necrotic centre represents the in vivo condition of a tumour. Here we have used both monolayers and spheroid form of the cancer cell line to assess the anti-cancer effect of the test drug (TTCC blocker), simultaneously differentiating the two modes of study. We hypothesise that the test drug would have an anti-cancer or an adjuvant effect in the cancer cell line; meanwhile, the results in spheroids would be more reliable compared to monolayer cell culture. We found that there was an increase in cell viability in monolayer culture after an appropriate drug dose, while in spheroids, there was a dose-dependent decrease in viability. In live/dead assay, in monolayer culture, PI stained cells were seen mostly in combined treatment while in spheroids, all the treated samples showed increased PI staining with a little damage at the surface. H&E staining showed that in monolayer, there was a morphology change only in combined treatment while in treated spheroid sections, there was an increase in the intercellular spaces. Migration assay showed that in monolayer all the treated samples showed reduced wound healing while in spheroids only in combined treatment there was a decrease in spheroid invasion in the extracellular matrix (ECM). We also tested the expression level of P-glycoprotein (Pgp), a protein responsible for cell-resistance by removing the drugs from the intracellular region, along with the expression of the isoforms of TTCC (Cav3.1, Cav3.2 and Cav3.3) by qPCR. We found that with the treatment, there was a reduced expression of both Cav3.1 and Cav3.2 while expression of Pgp increased and Cav3.3 expression was affected comparatively less than Cav3.1 and Cav3.2. With this, we conclude that the test drug showed a possible role as an anti-cancer as well as an adjuvant and the results in spheroid culture were more reliable. Though monolayer cell culture also showed the anti-cancer activity of the test drug, there was no uniformity of the results plus choosing the desired images from the N number of images taken from a sample can show the biased or manipulated selection of the results. Also, in monolayer cells, during live/dead assay there is always a possibility of losing the dead cells during the washing steps thus giving false results.